Formulations and methods of use of 2,2&#39;-dithio-bis-ethane sulfonate

ABSTRACT

This invention describes novel formulations containing a water soluble disulfide, 2,2&#39;-dithio-bis-ethane sulfonate, with or without cis-diammine dichloro platinum present in the same formulation, wherein the parenteral or oral administration of 2,2&#39;-dithio-bis-ethane sulfonate is used to reduce the risk or prevent or retard the development of cisplatin induced nephrotoxicity, myelosuppression, and neurotoxicity, and wherein the parenteral or oral administration of 2,2&#39;-dithio-bis-ethane sulfonate potentiates the antitumor activity of cisplatin when treating human patients with cancer. This invention also teaches novel formulations containing 2,2&#39;-dithio-bis-ethane sulfonate alone or in combination with cisplatin in lyophilized or dissolved in an aqueous formulations which can be administered to patients with cancer who are being treated with cisplatin. The invention also teaches methods of preparing said formulations and their use in preventing cisplatin related toxicities and potentiation of cisplatin antitumor activity.

This Application is a division of application number 08/553,005, filedNov. 3, 1995, pending which is a continuation-in-part of U.S. patentapplication Ser. No. 08/338,379 filed on Nov. 14, 1994, now U.S. Pat.No. 5,789,000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention teaches novel compositions and methods of use thereof, ofa water soluble disulfide, 2,2'-dithio-bis-ethane sulfonate (also knownas "disodium 2,2'-dithio-bis-ethane sulfonate", "dimesna" or "BNP7787")in human patients who are being treated for cancer with cis-diamminedichloro platinum (also known as "cisplatin" or "CDDP") and whereincis-diammine dichloro platinum and 2,2'-dithio-bis-ethane sulfonatecompositions are administered prior to, simultaneously or following theadministration of cisplatin to reduce the risk of cisplatin inducednephrotoxicity when treating human patients with cancer. This inventionalso teaches the use of said compositions containing2,2'-dithio-bis-ethane sulfonate as a key ingredient for the purposes ofpotentiating the antitumor activity of cisplatin in human subjects withcancer, and protecting against cisplatin related neurotoxicity andmyelosuppression. In its preferred aspect, this invention involves thepreparation and administration of a sterile, aqueous composition of2,2'-dithio-bis-ethane sulfonate to human patients with cancer who arebeing treated with cisplatin. In another preferred form, this inventioninvolves the preparation and administration of a lyophilized compositionof 2,2'-dithio-bis-ethane sulfonate, which is reconstituted with aqueousmedia prior to administration to human patients with cancer who arebeing treated with cisplatin. This invention also teaches methods ofadministration of the claimed 2,2'-dithio-bis-ethane sulfonatecompositions lacking cisplatin that can be carried out within 24 hourspreceding and within 24 hours following the administration of cisplatin.Yet another aspect of this invention is the methods of simultaneousadministration of cis-diammine dichloro platinum and2,2'-dithio-bis-ethane sulfonate wherein the 2,2'-dithio-bis-ethanesulfonate is administered simultaneously with the administration ofcis-diammine dichloro platinum. This simultaneous administration can becarried out by administering aqueous or lyophilized and reconstitutedcompositions containing 2,2'-dithio-bis-ethane sulfonate and cisplatinor by the simultaneous administration of each drug via separate routesof administration. Another aspect of this invention is a method ofreducing cis-diammine dichloro platinum induced nephrotoxicity,neurotoxicity, and myelosuppression and also potentiating cisplatinantitumor activity in human patients whose cancer is optimally treatedwith cis-diammine dichloro platinum.

This invention describes novel aqueous and lyophilized compositions andmethods of use thereof of a water soluble disulfide,2,2'-dithio-bis-ethane sulfonate and disodium 2,2'-dithio-bis-ethanesulfonate, which has been discovered by the inventors to protect againstcisplatin induced nephrotoxicity. The inventors also teach the use of2,2'-dithio-bis-ethane sulfonate as a key ingredient of the claimedcompositions for the purposes of preventing or retarding the developmentof cisplatin induced neurotoxicity and myelosuppression. The inventorshave made the unexpected discovery that 2,2'-dithio-bis-ethane sulfonatealso appears to potentiate (increase) the antitumor activity ofcisplatin in vivo and accordingly claim the use of2,2'-dithio-bis-ethane sulfonate for the purpose of increasing theantitumor activity of cisplatin. The novel lyophilized and aqueouscompositions contain 2,2'-dithio-bis-ethane sulfonate or disodium2,2'-dithio-bis-ethane sulfonate, with or without cis-diammine dichloroplatinum and the invention teaches that 2,2'-dithio-bis-ethanesulfonate, and pharmaceutically acceptable salts thereof, may beadministered simultaneously with or separately from cisplatin to reducethe risk of cisplatin induced toxicities when treating human patientswith cancer.

2. Description of the Related Art

A. Introduction

The use of cytotoxic anticancer drugs pose an increased risk of certaindrug related toxic side effects in human subjects undergoing cancertreatment. Drug toxicity associated with the use of anticancer drugsgreatly limits their clinical utility and safety in human subjects. Forexample, drug-induced impairment of cellular and/or organ functions mayresult in organ-specific toxicities in human subjects being treated forcancer. Additionally, the drugs themselves or their metabolites mayaccumulate or damage certain cellular components or impair certainbiochemical reactions in specific organs. The toxicities observed due tothe administration of anticancer drugs are usually dose dependent (e.g.,busulfan induced myelosuppression), are often related to cumulativedosages administered (e.g., BCNU induced pulmonary toxicity;anthracycline induced cardiac toxicity), and idiosyncratic drugtoxicities are noted with some frequency with certain anticancer drugs(e.g., mitomycin-C induced hemolytic uremic syndrome). By impairing ordamaging normal cellular function in specific organs the anticancer drugis causally connected with the drug-induced organ damage.

As a result of drug induced toxicity associated with the administrationof anticancer drugs to humans, clinicians attempt to prevent or reducethe risk of drug toxicity by certain pharmacologic maneuvers. Suchclinical maneuvers can impose risk of additional side effects, or resultin a dose reduction of the anticancer drug which in turn may adverselyaffect the likelihood of achieving control of the patient's tumor. Ifthe major dose-limiting organ toxicity of a particular anticancer drugis eliminated or substantially reduced, the result is that the safetyand efficacy of the primary anticancer drug is greatly increased. Asignificant reduction in drug toxicity in cancer treatment generallyresults in greater ability to administer higher doses of the drug,prevents or reduces the number of treatment delays, and increases thesafety and the quality of life for patients. An example of this approachis the use of G-CSF to reduce the duration and magnitude of drug inducedmyelosuppression resulting from the administration of several differenttypes of anticancer drugs. Therefore, an important area of drug researchand treatment is aimed at developing new methods to prevent or reducedrug induced dose limiting toxicities in human cancer patients.

This invention teaches new art which uses 2,2'-dithio-bis-ethanesulfonate or pharmaceutically acceptable salts thereof (includingdisodium 2,2'-dithio-bis-ethane sulfonate) contained in compositionswhich can be administered to human subjects with cancer being treatedwith cis-diammine dichloro platinum. For the purposes hereof, the usefulcomposition of matter defined by this invention includes2,2'-dithio-bis-ethane sulfonate, pharmaceutically acceptable salts of2,2'-dithio-bis-ethane sulfonate, dimesna and BNP7787. The inventorshave made an unexpected discovery that the use of compositionscontaining 2,2'-dithio-bis-ethane sulfonate are effective and safe inprotecting against cis-diammine dichloro platinum induced nephrotoxicity(impairment of normal renal function). The inventors have alsodiscovered that the administration of 2,2'-dithio-bis-ethane sulfonatecompositions is safe and non-toxic and appears to provide protectionagainst cisplatin induced myelosuppression, neurotoxicity and furtherappears to potentiate the antitumor activity of cisplatin.Myelosuppression is defined as the suppression of the production ofblood cells from the bone marrow. Cisplatin neurotoxicity can manifestas the impairment of peripheral sensory neural function (paresthesias,numbness, pain), impairment of central neural function (includingnausea, vomiting, ototoxicity, cranial nerve and ocular toxicities. Forthe purposes of this invention, cis-diammine dichloro platinum is alsoreferred to as "cisplatin" and "CDDP" interchangeably and withoutrestriction. The instant invention which encompasses novel compositionsand methods of use of 2,2'-dithio-bis-ethane sulfonate has tremendousutility in preventing, retarding the development of and reducing therisk of these cisplatin induced toxicities in human patients withcancer. This invention also teaches methods of manufacture of saidcompositions in the instant invention containing the water solubledisulfide 2,2'-dithio-bis-ethane sulfonate (or pharmaceuticallyacceptable salts thereof) alone or in combination with othermedicaments, when desired and add additional utility to this invention.

B. Cis-Diammine Dichloro Platinum

(1) Background of Cis-Diammine Dichloro Platinum

Cis-diammine dichloro platinum (referred herein to as "cisplatin" or"CDDP") is a widely used anticancer drug which is used in combinationwith other anticancer drugs in the treatment of cancers of the lung,breast, head and neck, ovary, esophagus, bladder, and testis. Along withits potent anticancer properties, cisplatin also has demonstratedclinically significant toxicities which limit its clinical utility andpose certain serious risks to patients undergoing treatment for cancer.The therapeutic benefits of cisplatin must always be carefully weighedagainst the possibility of these significant drug related toxicitiesassociated with its use. It is well known that one of the most importantand common dose limiting toxicities of cis-diammine dichloro platinum isrenal damage in patients receiving this drug for treatment of theircancer. Neurotoxicity and myelosuppression are also important toxicitiesrelating to cisplatin therapy in human patients (Perry, M. C., (1992)The Chemotherapy Source Book, Williams and Wilkins, 1172 pp.).

A stable and sterile aqueous solution of cisplatin in a sealed ampouleor vial containing a unit dosage form suitable for intravenousadministration to a human patient with cancer was described in U.S. Pat.No. 4,310,515, entitled "Pharmaceutical Compositions of Cisplatin"(Issued Jan. 12, 1982). The patent claims cisplatin provided in aconcentration between about 0.1 and about 1.0 mg/ml and a pH in a rangeof 2.0 to 3.0. The sterile aqueous cisplatin solutions may also containsodium chloride and mannitol. The present invention claims aqueouscompositions containing 2,2'-dithio-bis-ethane sulfonate alone or withany of the following components, in any combination without restriction:(a) sterile water for injection, (2) saline solutions with NaClconcentrations of about 0.1 to about 2.5%, (c) cisplatin in aconcentration of about 0.1 to 1.0 mg/ml, (d) sufficient quantities offormulation hydrochloric acid or phosphoric acid to maintain the pH ofsaid formulation in a pH range of about 2.0 to about 6.0, (e)pharmaceutical buffers such as sodium acetate or phosphate, and (f)mannitol in a concentration of about 1.0 to about 2.5%. The presentinvention also claims lyophilized formulations containing2,2'-dithio-bis-ethane sulfonate and pharmaceutically acceptable saltsthereof alone or in combination with any of the following withoutrestriction: (a) cisplatin, (b) NaCl, (c) mannitol, (d) phosphoric acid,and (e) pharmaceutical buffers such as sodium acetate or phosphate. Thepresent invention claims compositions and methods of use of saidcompositions which contain 2,2'-dithio-bis-ethane sulfonate inlyophilized or aqueous compositions that can be prepared andadministered before, during or after the administration of cisplatin tohuman subjects with cancer.

(2) Mechanisms of Action of Cis-Diammine Dichloro Platinum

Cisplatin exchanges chloride ions for nucleophilic groups such as RS-,R-S-CH₃, imidazole nitrogens and R-NH₂ to form linkages which can bevery stable. In an aqueous solution, one or both chloride ions may bereplaced by water to produce a hydrated intermediate known as an "aquocisplatin" species (See Reactions 1 and 2 below). The water molecule(s)attached to the cisplatin can be subsequently eliminated by an incomingnucleophile. In some cases there can be direct displacement of thechloride ion by an incoming nucleophile without the participation of thesolvent. Thus, several species of cisplatin ("Pt") exist in solution asdefined according to the following equilibria:

Reaction 1 Pt(NH₃)₂ Cl₂ +H₂ O-> Pt(NH₃)₂ Cl(H₂ O)!⁺

Reaction 2 Pt(NH₃)₂ Cl(H₂ O)!⁺ +H₂ O-> Pt(NH₃)₂ (H₂ O)₂ !⁺⁺

The addition of chloride ions to the medium shifts the equilibrium tothe left and the reactivity of the cisplatin species depends on thechloride concentration of the medium. Isotonic and hypertonic salinesolutions have high chloride ion concentrations and cisplatin willpredominate as the Pt(NH₃)₂ Cl₂ species. The displacement of chlorideions from Pt(NH₃)₂ Cl₂ in an environment with a high chlorideconcentration occurs very slowly over time with exposure to thestrongest nucleophiles, such as the sulfur anion. For example,formulation of cisplatin with sodium thiosulfate or 2-mercapto ethanesulfonate (mesna) is impractical because of the chemical quenching ofcisplatin with the sulfur anion or the sulfhydryl moiety of thiosulfateand mesna, respectively. Cisplatin is also directly incompatible withdiethyldithiocarbamate due to the presence of the sulfur anion, and thuscannot be formulated in the same solution for parenteral administration.

Cisplatin is believed to act on tumor cell DNA by forming intrastrandcrosslinks of the drug attaching to the N7 atom of the imidazole betweenadjacent purine bases comprising predominantly sequences of 5'-GG-3',5'-AG-3', 5'-GA-3' or 5'-GXG-3' where X is a naturally occurring purineor a pyrimidine (e.g., adenine, thymine, cytosine or guanine)(Eastman,A., Biochemistry, 25, 3912-3915, 1986; Pinto, A. L. and Lippard, S.,Biochem. Biophy. Acta, 780, 167-180, 1986). Cisplatin is believed toexert its antitumor effects by the formation of intrastrand crosslinkswhich may result in alterations in DNA structure or function. In orderfor cisplatin to react with certain nucleic acid sequences in cellularDNA, it must first undergo chemical conversion to an active species bythe displacement of chloride ligands with water to form the mono-aquo ordi-aquo species. The aquo species of cisplatin is reactive withnucleophilic species, including the imidazole nitrogens on DNA orsulfhydryl groups which are also present in cells forming the renaltubular epithelium in humans.

Cisplatin readily reacts with compounds containing sulfhydryl moieties.Sulfhydryl groups are found in cysteine, glutathione, and homocysteine.Metallothionein is a 7 kDa protein which has a high (approximately 30%)cysteine content Kelly, S. L. et al., Science, 241, 1813-1815, 1988).Increased cellular concentrations of metallothionein and glutathionehave been correlated with drug resistance to cisplatin therapy. Thus, ifthe local renal tubular concentration of sulfhydryl groups from2-mercapto ethane sulfonate is increased, then cisplatin toxicity may bereduced by the chemical quenching of the cisplatin aquo species in therenal tubules. The present invention accomplishes this objective.

The claimed invention teaches a new discovery in direct contrast topreviously reported views of the pharmacology and metabolism of2-mercapto ethane sulfonate (mesna). For the purposes of this invention,the administration of 2,2'-dithio-bis-ethane sulfonate preceding,following or simultaneously with cisplatin administration provides aphysiologically safe source of additional sulfhydryl groups in theproximal and distal convoluted tubules to prevent renal toxicity. Unlikemesna, 2,2'-dithio-bis-ethane sulfonate is chemically inert with respectto cis-diammine dichloro platinum, and thus is compatible in the claimedformulation.

In the present invention, the chemical and pharmacologic behavior ofparenterally or orally administered 2,2'-dithio-bis-ethane sulfonate issubstantially different from the parenteral or oral administration ofmesna as follows:

1. The 2,2'-dithio-bis-ethane sulfonate contained in the compositionremains largely intact in the plasma;

2. 2,2'-dithio-bis-ethane sulfonate is a dianionic species and enterscells to a much lesser degree than mesna-cysteine, mesna-glutathione,and mesna-homocysteine disulfide conjugates. Therefore, more disulfideis filtered and excreted via the renal route, making more thiolsavailable for detoxification in the kidney relative to parenterally ororally administered mesna; and

3. In the kidney, 2,2'-dithio-bis-ethane sulfonate undergoes reductionby renal glutathione reductase and thiol transferase enzymes to formfree 2-mercapto ethane sulfonate, which in turn reacts with aquo speciesof cisplatin resulting in renal excretion of non-toxic cisplatin-2-mercapto ethane sulfonate conjugates.

For the purposes of this invention, the simultaneous or separateadministration of cisplatin with 2,2'-dithio-bis-ethane sulfonate willprovide a physiologically safe source of additional sulfhydryl groups inthe proximal and distal convoluted tubules to prevent renal toxicity.Unlike mesna, 2,2'-dithio-bis-ethane sulfonate is chemically inert underproper conditions with respect to cis-diammine dichloro platinum, andthus is compatible in claimed formulations, and additionally whenconvenient, 2,2'-dithio-bis-ethane sulfonate can be administeredseparately preceding or following the administration of cisplatin asclaimed in this invention.

It has been previously reported that oral or parenteral administrationof 2-mercapto ethane sulfonate (mesna) to mice, rats or humans resultsin spontaneous autoxidation of mesna to form 2,2'-dithio-bis-ethanesulfonate (dimesna) in the plasma. James and Rogers reported an HPLCassay for plasma thiols using an electrochemical detector (James, C. A.and Rogers, Journal of Chromatography, 382, 394-398, 1986). Theinventors submit that by using the method of James and Rogers, thedetection and chemical characterization of dimesna (the putative humanplasma metabolite of mesna) is indirect because this method can notchemically distinguish between dimesna, mesna-cysteine, andmesna-homocysteine conjugates. The HPLC method of James and Rogersinvolved the reduction of the samples by sodium borohydride and thesamples were subsequently assayed for thiols. The difference inconcentration from the unreacted initial sample and the sample that hadbeen reacted with sodium borohydride was used to determine the amount ofwhat was thought to be dimesna in the plasma. The inventors contend thatsodium borohydride would react with mesna-mesna, mesna-cysteine,mesna-glutathione, mesna-homocysteine conjugates and thus, they submitthat the method of James and Rogers fails to distinguish and quantitatethe relative amounts of these entities which would form as a consequenceof mesna metabolism. Other investigators rely on a similar or identicalmethod as used by James and Rogers. All of these reports fail to mentionor take into account the possibility of mesna forming a significantproportion of thiol conjugates with entities other than mesna, e.g.,mesna-cysteine, mesna-glutathione, mesna-homocysteine. The inventorssubmit that the method of James and Rogers is ambiguous, imprecise andnot capable of specifically identifying the disulfide conjugate formed.

The inventors also note that increased cysteine elimination in the urinehas been reported in association with the administration of mesna tohuman subjects (Sidau, B. and Shaw, I. C., J. Chromatography, 311,234-238, 1984). This observation indirectly supports the inventors'current hypothesis of disulfide conjugation of mesna with other thiolsin the plasma (FIG. 1, "New Hypothesis", Middle Column). The enhancedcysteine elimination in urine, as reported by Sidau et al., can beexplained by the current invention. The inventors contend that adisulfide linkage forms between mesna and cysteine in the plasma andreduction by glutathione reductase and thiol transferase of themesna-cysteine conjugates occurs in the renal tubular system to generatefree thiols. The free thiols are predicted to react with toxic aquometabolites of cisplatin.

In view of the above discussion, the inventors submit that theconcurrent, pre- or post-administration of 2,2'-dithio-bis-ethanesulfonate with cisplatin is chemically and pharmacologically superior tousing mesna because: (1) a greater amount of disulfides will bedelivered to the renal tubular system whereupon these disulfides areavailable for reduction by glutathione reductase and thiol transferasesto form the free thiol, 2-mercapto ethane sulfonate, and (2) a loweramount of energy is needed to reduce the 2,2'-dithio-bis-ethanesulfonate disulfide linkage which in turn will generate a greater amountof free thiols in the renal tubules whereupon these free thiols canreact with the toxic aquo species of cisplatin.

An object of this invention is the simultaneous or separate, parenteraladministration of 2,2'-dithio-bis-ethane sulfonate and cisplatin tohuman subjects with cancer. Another object of the present invention isthe separate administration of 2,2'-dithio-bis-ethane sulfonatepreceding or following the administration of cisplatin. Another objectof this invention is oral or parenteral administration of2,2'-dithio-bis-ethane sulfonate to human patients with cancer. Yetanother object of this invention is the administration of2,2'-dithio-bis-ethane sulfonate for the purpose of preventing, reducingor retarding the development of cisplatin related neurotoxicity,myelosuppression and the use of 2,2'-dithio-bis-ethane sulfonate for thepurposes of potentiating (increasing) the antitumor activity ofcisplatin in human subjects with cancer. The following characteristicsof 2,2'-dithio-bis-ethane sulfonate support its use in the presentinvention:

1. 2,2'-dithio-bis-ethane sulfonate will predominate in the plasmacompartment.

2. 2,2'-dithio-bis-ethane sulfonate is an anionic species because it hastwo negatively charged oxygens. Because of this anionic characteristic,the molecule penetrates cell membranes, especially those of cancercells, very poorly.

3. The highly anionic nature and small molecular size of2,2'-dithio-bis-ethane sulfonate are key properties which account forits rapid and nearly exclusive excretion in high concentrations throughthe kidney.

4. In the absence of any other treatment, 2,2'-dithio-bis-ethanesulfonate has reportedly been tested only once in normal humanvolunteers (Shaw, I. C. and Weeks, M. S., Eur J Cancer Clin Oncology23:933-935; 1987; Brock N., et al., J. Cancer Res. Clin. Oncol., 108,87-97, 1984; Brock N., et al., Eur. J. Cancer Clin. Oncol. 18,1377-1387, 1982; Brock, N. et al., Eur. J. Cancer Clin. Oncol. 17,1155-1163, 1981). However, the investigators in that instance failed toconfirm the chemical identity of the metabolites in the plasma and urineof the human subjects. 2,2'-dithio-bis-ethane sulfonate has not beenadministered to human subjects being treated with platinum basedtherapies.

Cisplatin induced nephrotoxicity may be reduced because of the highlocal concentration of 2-mercapto ethane sulfonate that may be generatedfrom 2,2'-dithio-bis-ethane sulfonate by renal tubular enzymes such asgamma glutamyl transpeptidase and thiol transferases in the same regionof the renal tubules where the formation or delivery of a highconcentration of aquo species of cisplatin is achieved. If cisplatin isconjugated with glutathione or other free thiols, these cisplatinconjugates are likely to be nephrotoxic if further metabolized tomercapturic acids (Hanigan M. H. et al. Cancer Research 54:5925; 1995).2,2'-dithio-bis-ethane sulfonate, or its metabolite 2-mercapto ethanesulfonate, may prevent conjugation of cisplatin with glutathione orother endogenous thiols and/or further metabolism of cisplatinconjugates to mercapturic acids which are believed to be nephrotoxic.

This invention is also useful because the simultaneous or separate oralor parenteral administration of 2,2'-dithio-bis-ethane sulfonate andcisplatin: (1) insures treatment compliance, (2) will reduce pharmacypreparation costs, (3) will reduce errors in prescribing both drugs, (4)will reduce the amount of additional prophylactic maneuvers needed inorder to reduce toxicity and avoid iatrogenic related complications(i.e. furosemide, or hypertonic saline administration as describedabove), and (5) for greater utility when a longer shelf life is desired,this invention also teaches methods to make and use lyophilizedformulations containing 2,2'-dithio-bis-ethane sulfonate alone or incombination with cisplatin, mannitol, buffers and sodium chloride.2,2'-dithio-bis-ethane sulfonate can be contained in a lyophilizedformulations with other useful medicaments such as cisplatin, sodiumchloride, mannitol, and buffers in any combination and withoutrestriction.

Formulations of cisplatin and 2,2'-dithio-bis-ethane sulfonate must bemaintained at a pH less than 7.0 and greater than 1.0 because of theneed to prevent the formation of aquo species of cisplatin and also toprevent the formation of mesna which could subsequently react withcisplatin species. Another important component of this invention is theuse of sufficiently high concentrations of NaCl (e.g., 0.9% or greater)and HCl because the stability of cisplatin is proportionally related tothe chloride ion concentration of the solution.

C. Nephrotoxicity Associated with Cis-Diammine Dichloro PlatinumAdministration

One of the most important limitations in the human clinical use ofcisplatin is the nephrotoxicity which develops as a consequence ofcumulative and dose dependent exposure to the drug, or which may occurin setting the administration of cisplatin to patients with renalinsufficiency or co-administration of other nephrotoxic agents (e.g.,aminoglycosides) (Rozencweig et al., 1977, Cis-diamminedichloroplatinum(II), Ann. Intern. Med., 86, 803; Gonzalez-Vitale et al., 1977, Therenal pathology in clinical trials of cisplatin (II) diamminedichloride,Cancer, 39, 1362; Campbell et al., 1983, Plasma platinum levels:Relationship to cisplatin dose and nephrotoxicity, Cancer Treat. Rep.,67, 169; Offerman et al., 1984, Acute effects ofcis-diammine-dichloroplatinum (CDDP) on renal function, CancerChemother. Pharmacol, 12, 36).

The major clinical features of cisplatin induced nephrotoxicity includedecreases in creatinine clearance, elevated creatinine, elevated BUN,elevated uric acid and hypomagnesemia. The dose limiting toxicity ofcisplatin when administered as a single dose per cycle isnephrotoxicity. Nephrotoxicity associated with cisplatin administrationmay also be related to the peak plasma concentration of the drug.Hyperuricemia and hypoalbuminemia are predisposing factors to cisplatinnephrotoxicity along with renal insufficiency, concomitantadministration of other drugs, including aminoglycoside antibiotics andpossibly by amphotericin B.

Typical pathological changes in the kidneys after cisplatin applicationhave been observed in laboratory animals and humans. (Kociba andSleight, 1971, Acute toxicologic and pathologic effects of cis-diamminedichloro platinum in the male rat, Cancer Chemother. Rep., 55, 1; Choieet al., 1981, Acute and chronic cisplatin nephropathy in rats, Lab.Invest., 44, 397; Goldstein and Gilbert, 1983, The nephrotoxicity ofcisplatin, Life Sci., 32, 685). Certain strains of rats have been notedfor their excellent correlation with human nephrotoxicity, includingFischer, Wistar, and Harlan-Sprague Dawley rats. These cisplatin inducedrenal lesions are dose and time dependent, and are mainly localized inthe outer stripe of the medulla of the kidney, which corresponds to themicroscopic anatomic location of the glomerulus and convoluted tubules.

Thus, cisplatin induced nephrotoxicity usually occurs as a result of thecumulative and dose dependent exposure to the drug, or when administeredto patients with renal insufficiency or when coadministered with anothernephrotoxic agent (e.g., aminoglycosides). The dose limiting toxicity ofcisplatin, when the drug is administered as a single dose per cycle, isnephrotoxicity which may be related to the peak plasma concentration ofthe drug itself.

Cisplatin induced nephrotoxicity is a clinically important problemassociated with the use of the drug, and certain clinical maneuvers aregenerally employed in an attempt to reduce the risk of thiscomplication. These prophylactic maneuvers include:

a. Parenteral administration of hypertonic (3%) NaCl; (Ozols et al.,1984, High-dose cisplatin in hypertonic saline, Ann. Intern. Med., 100,19);

b. Parenteral administration of normal (0.9%) NaCl;

c. Mannitol diuresis (Hayes et al., 1977, High dose cisplatin diamminedichloride, amelioration of renal toxicity by mannitol diuresis, Cancer,39, 1372);

d. Pre- and/or post treatment hydration (oral or parenteral);

e. Forced diuresis by the administration of loop diuretics such asfurosemide (Ostrow et al., 1981, High-dose cisplatin therapy usingmannitol versus furosemide diuresis: comparative pharmacokinetics andtoxicity, Cancer Treat. Rep., 65, 73); and

f. Oral or parenteral administration of reduced thiols such asdiethyldithiocarbamate (rodents), thiosulfate (humans), or 3-aminopropylamino ethylphosphorothioic acid (WR-2721).

However, these maneuvers have certain drawbacks which limit thepractical use of cisplatin and introduce additional definite risks fortreatment related complications in patients undergoing treatment. Forexample, the administration of hypertonic saline (NaCl 3.0%) poses therisk of iatrogenic hypernatremia. Hypernatremia is a life threateningmedical emergency which can be fatal, and the administration ofhypertonic saline is contraindicated in patients with elevated serumsodium or patients with congestive heart failure. The administration ofnormal saline (NaCl 0.9%) in patients increases the risk of fluidoverload in patients. The use of powerful loop diuretics to increaseurine production by the kidney such as furosemide increase theiatrogenic risk of hypokalemia, hyponatremia, hypocalcemia, hypovolemia,metabolic alkalosis and hypochloremia. All of these conditions can belife threatening and in some cases are fatal.

It is important to note that these maneuvers aimed at prophylaxis ofcisplatin nephrotoxicity require additional clinical services,additional patient monitoring (e.g., physicians, nurses, andpharmacists), and additional hospitalization expense. Additionally,since these prophylactic maneuvers (aimed at reducing the risk ofnephrotoxicity) are separate from the administration of the drug(cis-diammine dichloro platinum), the patient runs the risk ofexperiencing additional toxicity due to the maneuver itself (e.g., fluidoverload, congestive heart failure, hyperosmotic state, hypernatremia orby physician, nurse, pharmacist or support staff human error).

This invention reduces cisplatin induced nephrotoxicity, neurotoxicityand myelosuppression and potentiates the antitumor activity of cisplatinby the oral or parenteral administration of 2,2'-dithio-bis-ethanesulfonate to human subjects being treated with cisplatin for cancertherapy. The 2,2'-dithio-bis-ethane sulfonate and cis-diammine dichloroplatinum are in compositions suitable for administration to humansubjects with cancer, or alternatively 2,2'-dithio-bis-ethane sulfonateis administered separately from cisplatin. As discussed above,parenteral formulations of mesna (mercapto ethane sulfonate sodium) orsodium thiosulfate with cisplatin are not practical because thesulfhydryl groups on mesna or the sulfate anion of sodium thiosulfatewill react with cisplatin yielding inactive species of cisplatin.

D. Water Soluble Thiols as Detoxifying Agents in the Kidney

1. 2-Mercaptoethane Sulfonate Sodium or "Mesna"

Mesna is a pharmacologically safe thiol that has been used clinically inhuman subjects for approximately two decades. Mesna has been reported tobe rapidly eliminated through the kidneys, accumulates in the urine and,unlike cysteine or N-acetyl cysteine, only slightly penetrates cellularmembranes. In the rat, over 80% of the administered dose of mesna isreportedly recovered in the urine within three hours after intravenousadministration (Pohl et al., Meth. Find. Clin. Pharmacol. 3(Suppl 1),95-101, 1981).

For the purposes of the present invention, the inventors wish to pointout that the analytical methods previously used to detect the presenceof mesna metabolite in the plasma or in the urine in the studies areincapable of determining the chemical identity of the thiol (seediscussion above). Mesna is widely used to reduce or prevent the risk ofhemorrhagic cystitis to the uroepithilium which is associated with theuse of chloroethylnitrosoureas (including BCNU, CCNU and MeCCNU) andcertain oxazaphosphorine type anticancer drugs which includecyclophosphamide, ifosfamide and trophosphamide. Mesna administeredorally or parenterally to human subjects significantly reduces theincidence of uroepithelial toxicity in patients receiving therapy withthese drugs. Oxazaphosphorine induced hemorrhagic cystitis can be a lifethreatening condition due to profuse bleeding from the uroepithelialsurfaces involving the ureters, bladder and urethra.

It is especially important to note for the present invention thatoxazaphosphorine or chloroethylating agent induced uroepithelialtoxicity is chemically, biochemically, anatomically and pathologicallydistinct from the renal toxicity which is observed with administrationof cisplatin. Cisplatin is an inorganic molecule whereasoxazaphosphorines and chloroethylating type anticancer drugs are organicmolecules. The toxic species of cisplatin is the inorganic mono- anddi-aquo species whereas the toxic species of oxazaphosphorines is theorganic molecule, acrolein and for chloroethylating drugs are thechloroethyl intermediates. The toxic species of oxazaphosphorines andchloroethylating anticancer drugs are chemically distinct and resultfrom entirely different precursors and produce damage in tissues whichis clearly different from that of cisplatin and its metabolites.Cisplatin damages renal tubular cells whereas oxazaphosphorines andchloroethylating type anticancer drugs damage the uroepithelium (renalpelvis, ureters, bladder and urethra). It is also important to note thatthe organic chemical interactions of mesna with acrolein, the toxicspecies produced by the metabolism of anticancer oxazaphosphorines whichdirectly damages the uroepithelium, is entirely different than theproposed inorganic chemical interactions which lead to detoxification ofcisplatin by mesna.

Dimesna, the only reported metabolite of mesna, is reportedly formedspontaneously by autoxidation of mesna in the plasma. Mesna dimers (twomesna molecules covalently attached via a disulfide linkage) arereported to predominate in the blood following mesna administration.This mesna dimer metabolite is reportedly eliminated through the kidneysby glomerular filtration, being partly reduced to mesna during excretion(Brock, et al., Arzneim Forsch, 32, 486-487, 1982). Reportedly, anaverage of approximately 45% of the administered mesna dose is found inthe urine in the form of mesna, the reactive thiol. The remainder foundis reportedly a mesna metabolite, dimesna.

2. Bioavailability of Orally Administered Mesna In 1984, Burkert et al.described the bioavailability of orally administered mesna (sodium2-mercaptoethane sulfonate, Uromitexan; Burkert et al.,Arzneim.-Forsch./Drug Res. 34, 1597, 1984). Previous experimentalstudies in rats had demonstrated that mesna was absorbed from theintestine following oral administration and that it passed unchangedthrough the hepatic vascular system (Brock et al., J. Cancer Res. Clin.Oncol. 108, 87 1984; Ormstad et al., Cancer Research, 43, 333, 1983). Itwas proposed that in the plasma mesna was rapidly oxidized to disulfidedimesna and that the reaction occurs when mesna was injectedintravenously. It was further proposed that after glomerular filtration,about 50% of dimesna was reduced to mesna in the renal tubularepithelium.

The inventors propose that it is unlikely that oral or parenterallyadministered mesna would necessarily be oxidized in the plasma to formsignificant quantities of dimesna (e.g., quantities greater than 40% ofan administered dose). The inventors propose that the significantmajority of mesna reacts with other plasma thiols to form conjugateswith cysteine, glutathione and other thiol containing amino acids whichare small enough to still undergo glomerular filtration and possiblytubular secretion and would be cleaved to form mesna (plus the freeamino acid) in the tubular lumen. The inventors also specificallypropose for the first time that mesna reacts predominantly with otherthiols in the plasma such as cysteine, homocysteine, or the cysteinecontained in glutathione (See above discussion). Since mesna forms adisulfide linkage with thiol containing amino acids or peptides it couldstill be filtered by the glomerulus or secreted in the proximal tubule.The therapeutic disadvantage of these mesna-cysteine and other similardisulfide conjugates is that their disulfide linkages are chemicallymore stable than the 2,2'-dithio-bis-ethane sulfonate conjugate proposedin the present invention, which results in a less facile chemicalproduction of free thiols in the kidney.

In their study, Burkert et al. (Burkert et al., Bioavailability oforally administered mesna, Arzneim.-Forsch./Drug Res. 34, 1597, 1984)confirmed the established bioavailability of orally administered mesnawith studies in healthy volunteers and patients with tumors. Burkert et.al. tested the oral administration of mesna (Uromitexan drink ampoules)in 18 healthy probands and in 5 tumor patients. Following a single oraladministration of either 20 or 40 mg/kg mesna, approximately 52% of thedose was excreted in the urine as reactive thiol groups and the onlymetabolite of mesna, mesna disulfide or dimesna, comprised the remaining48%. The experimental methods used to characterize mesna or dimesna inthese studies are not conclusive in establishing with certainty theprecise chemical identity of mesna or dimesna as urinary metabolites. Itis far more likely that mesna was conjugated with certain thiols such ascysteine, homocysteine or glutathione. The key description used tocharacterize the identity of the putative dimesna metabolite by previousresearch groups is "thiol groups" which could represent other aminoacids.

This study also concluded that after intravenous injection of 20 mg/kgmesna, about 48% of the dose administered appeared as thiol groups inthe urine. It took approximately 13 hours (20 mg/kg p.o.) or 18 hours(40 mg/kg p.o.) for the concentration to drop below the minimumconcentration presumed to still be protective (100 ug/ml). However, theelimination pattern and the time required to reach the thresholdconcentration of mesna varies dramatically from patient to patient.

3. Concomitant Use of Oral Mesna in Rats or Thiosulfate in Humans toReduce the Urotoxic Effects Of the Cisplatin

Kempf et al. studied the effects of per os administration of sodium2-mercaptoethane-sulfonate (mesna) in rats in preventing the nephrotoxiceffects of cisplatin administered intraperitoneally (Kempf et al.,Effective prevention of the nephrotoxicity of cisplatin (CDDP) byadministration of sodium 2-mercaptoethane-sulfonate (mesna) in rats, Br.J. Cancer, 52, 937-939, 1985). As described above, mesna is extensivelyused in patients who receive oxazaphosphorine antitumor drugs such ascyclophosphamide and ifosfamide to protect the urinary tract, especiallythe ureters and bladder, against the toxic organic metabolite, acrolein(Brock, et al., 1982). In the case of oxazaphosphorines, acrolein isproduced as a result of their metabolism and mesna undergoes addition tothe double bond of acrolein, resulting in a stable thioether adductwhich has no damaging effects on the uroepithelium and is excreted inthe urine.

For the purpose of this invention, it is important to distinguish thatthe acrolein metabolite, an organic molecule, of the oxazaphosphorinesis associated with uroepithelial toxicity, and in absolute contrast,cisplatin and its aquo species, which are inorganic molecules, areassociated with direct toxicity to the kidney (nephrotoxicity or renaltoxicity). Therefore, the uses of 2-mercapto ethane sulfonate as aprotective agent against oxazaphosphorine and chloroethylating agentassociated toxicities are entirely different from the protective uses of2,2'-dithio-bis-ethane sulfonate in the present invention.

Howell and colleagues (Howell et al., Intraperitoneal cisplatin withsystemic thiosulfate protection, Ann. Int. Med., 97, 845-851, 1982)administered thiosulfates by intravenous infusion to cancer patientsreceiving intraperitoneal cisplatin. They observed that much higher(more than two fold) doses of cisplatin per meter square could beadministered intraperitoneally and that renal toxicity could beprevented when thiosulfates are administered by the intravenous route.The inventors note that the use of sodium thiosulfate in an aqueousformulation of cis-diammine dichloro platinum is not practical becausethiosulfate will inactivate cisplatin and is incompatible in the samesolution.

Protection against the nephrotoxicity of intraperitoneally administeredcisplatin in rats through oral mesna administration has not beenestablished until the work of Kempf et al. As stated above, mesnadisulfide is the only reported metabolite of mesna and mesna disulfidedoes not readily react with electrophilic alkylating agents such asnitrogen mustard or oxazaphosphorines (Brock et al., The development ofmesna for the inhibition of urotoxic side effects of cyclophosphamide,ifosfamide, and other oxazaphosphorine anticancer drugs, Rec. Res.Cancer Res., 74, 270, 1980). After oral administration of mesna, theformation of dimesna reportedly occurs almost solely in the blood. Afteri.v. administration of mesna, the disulfide is reportedly spontaneouslyformed by autoxidation and found predominantly in the blood stream(Brock et al., Studies on the urotoxicity of oxazaphosphorinecytostatics and its prevention. Eur. J. Cancer. Clin. Oncol. 18, 1377,1982). Brock and co-workers reported that dimesna is eliminated throughthe kidneys by glomerular filtration, and, to a great extent, reduced tomesna during excretion.

Using per os administration of mesna, Kempf et al. demonstrated completeprevention of renal damage in rats after a single i.p. dose of 3 mgcisplatin /kg body weight. Their data demonstrated a clear dose/effectrelationship, in that low doses of mesna only partially protected thekidneys of rats from renal damage. The inventors wish to point out that2-mercapto ethane sulfonate differs vastly from 2,2'-dithio-bis-ethanesulfonate on a physicochemical, biochemical, toxicological andpharmacologic basis in terms of formulation compositions and methods ofuse claimed in the instant invention.

The study of Kempf et al. notably involved the intraperitonealadministration of cisplatin with prior and subsequent oraladministration of mesna in rats. The pharmacokinetics ofintraperitoneally administered cisplatin differ substantially from theparenteral (e.g., intravenous) administration of cisplatin. In the caseof intraperitoneal administration of cisplatin, it is possible toachieve much higher local (intraperitoneal) concentrations of cisplatin,and there is less risk of nephrotoxicity because intraperitonealcisplatin and its various species do not achieve similar peak plasmaconcentrations to those achieved with intravenous administration ofcisplatin. The oral administration of 2,2'-dithio-bis-ethane sulfonateis predicted by the inventors to be substantially different as follows:(a) intravenous or intraarterial administration of2,2'-dithio-bis-ethane sulfonate is predicted to reach higher plasmaconcentrations and greater area under the curve values at given dosagesrelative to orally administered 2,2'-dithio-bis-ethane sulfonate, and(b) the plasma half life of 2-mercapto ethane sulfonate versus2,2'-dithio-bis-ethane sulfonate will be different in humans. It is alsoimportant to note that at maximally tolerated dosages, the peak plasmaconcentration and the amount of cisplatin excreted by the kidney is lessduring intraperitoneal administration than the peak plasma concentrationand amount of cisplatin excreted by the kidney when cisplatin isadministered intravenously.

The experiments reported by Kempf et al. in rats completely fails totest or describe the ability of parenterally or orally administered2,2'-dithio-bis-ethane sulfonate in humans to protect against anytoxicities related to intravenously administered cisplatin. Theinventors point out that there is no art which teaches theadministration of 2,2'-dithio-bis-ethane sulfonate to humans with anytype of platinum anticancer drug. Because of the above discussion andthe literature, one could surmise that the simultaneous administrationof mesna would result in the inactivation of cisplatin. Mesna isdirectly incompatible with cisplatin and is reported in the Physician'sDesk Reference (p. 661 1994 Edition, Medical Economics Data ProductionCompany). The inventors have determined that the parenteraladministration of 2,2'-dithio-bis-ethane sulfonate and cisplatin canresult in an increase in cisplatin nephrotoxicity at lower doses of2,2'-dithio-bis-ethane sulfonate; this increase in toxicity can beovercome by administration of higher doses of 2,2'-dithio-bis-ethanesulfonate. This observation has not been reported before. Prior to thisinvention, one cannot predict that the simultaneous administration of2,2'-dithio-bis-ethane sulfonate would or would not result in cisplatininactivation or an increase in cisplatin toxicity. The inventors furtherbelieve that the administration of mesna does not result in theformation of substantial amounts of dimesna in the plasma; rather mesnapredominantly forms conjugates with other plasma thiols, especially theamino acid cysteine which is abundant in the plasma (see abovediscussion). Additionally, the inventors have determined that theadministration of 2,2'-dithio-bis-ethane sulfonate with cisplatinabrogates other cisplatin related toxicities, including myelosuppressionand neurotoxicity. The inventors have also made the unexpected discoverythat the administration of 2,2'-dithio-bis-ethane sulfonate canpotentiate the antitumor activity of cisplatin. The2,2'-dithio-bis-ethane sulfonate mediated abrogation of cisplatininduced myleosuppression and neurotoxicity, and the potentiation ofcisplatin antitumor activity are new discoveries and have not beenpreviously reported by others.

Thus, this invention is novel because: (1) novel methods of useinvolving the parenteral administration of cisplatin and oral orparenteral administration of 2,2'-dithio-bis-ethane sulfonate areclaimed, (2) methods of making and using compositions of2,2'-dithio-bis-ethane sulfonate alone or with cisplatin in sterileaqueous or lyophilized compositions wherein said compositions aresuitable for use in human patients with cancer are claimed, (3) thepresent invention describes the use of 2,2'-dithio-bis-ethane sulfonateas a key ingredient for the purpose of retarding or abrogating thedevelopment of cisplatin induced nephrotoxicity, myelosuppression andneurotoxicity, and (4) the present invention teaches that2,2'-dithio-bis-ethane sulfonate as a key ingredient can be used topotentiate the antitumor activity of cisplatin.

As stated above, this invention challenges the previous reports thatmesna, to a large extent, forms dimesna in the plasma of humans. Theinventors submit that oral or parenteral administration of mesna tohuman subjects results in the formation of more mesna-cysteineconjugates which are excreted by the kidney as compared to mesna-mesnaconjugates. Cysteine, homocysteine and glutathione are endogenous thiolsin human plasma and therefore could undergo reaction with mesna.

The inventors have also determined that the administration of2,2'-dithio-bis-ethane sulfonate with cisplatin can result in protectionagainst cisplatin induced myelosuppression and neurotoxicity, and haveobserved potentiation of the antitumor activity of cisplatin in tumorbearing animals. The inventors do not wish to be bound by anyhypothetical explanation of the possible mechanisms of these beneficialeffects of the administration of 2,2'-dithio-bis-ethane sulfonate inhumans treated with cisplatin. The inventors postulate that thebeneficial increase in antitumor activity in tumor bearing animals maybe the result of altered pharmacokinetic behavior of cisplatin in thepresence of 2,2'-dithio-bis-ethane sulfonate as a consequence of anincreased peak plasma concentration or area under the curve ofcisplatin, or a combination of the two pharmacokinetic parameters.Another hypothesis relating to the potentiation of the antitumoractivity of cisplatin is that 2,2'-dithio-bis-ethane sulfonate isforming a complex with cisplatin that results in altered pharmacokineticbehavior of cisplatin in the form of any of the following parameters:increased effective tumor uptake of cisplatin, increased peak plasmaconcentrations of cisplatin or increased area under the curve ofcisplatin.

SUMMARY OF THE INVENTION

2,2'-dithio-bis-ethane sulfonate, and pharmaceutically acceptable saltsthereof, is the key ingredient of all the claimed compositions and usesthereof in the present invention. Pharmaceutically acceptable salts of2,2'-dithio-bis-ethane sulfonate include: (a) disodium2,2'-dithio-bis-ethane sulfonate, (b) monosodium 2,2'-dithio-bis-ethanesulfonate, (c) sodium potassium 2,2'-dithio-bis-ethane sulfonate, (d)dipotassium 2,2'-dithio-bis-ethane sulfonate, (e) calcium2,2'-dithio-bis-ethane sulfonate, (f) magnesium 2,2'-dithio-bis-ethanesulfonate and (g) monopotassium 2,2'-dithio-bis-ethane sulfonate. Any ofthese salts may be used in the present invention, but the disodium,monosodium, calcium and magnesium salts are more preferred. When othersalts of 2,2'-dithio-bis-ethane sulfonate are used in place of thedisodium salt, the concentration or proportion of the sulfonate will beadjusted according, to molarity. 1 g of the disodium salt,2,2'-dithio-bis-ethane sulfonate is equivalent to 4.63 millimoles of2,2'-dithio-bis-ethane sulfonate anion. Hereinafter, the invention willbe discussed with reference to aqueous compositions of disodium2,2'-dithio-bis-ethane sulfonate, but the person skilled in the art willreadily be able to use the principles of this invention in relation toother compositions containing salts of 2,2'-dithio-bis-ethane sulfonateor its anionic form.

Compositions containing cisplatin and 2,2'-dithio-bis-ethane sulfonatemust be maintained at a pH less than 7.0 because of the need to preventor retard the rate of formation of appreciable quantities of the aquospecies of cisplatin and also to prevent or retard the formation of2-mercapto ethane sulfonate which could subsequently react withcisplatin species. Aqueous or lyophilized compositions that can bereconstituted with a pharmaceutically acceptable aqueous diluentcontaining 2,2'-dithio-bis-ethane sulfonate that do not containcisplatin can have a much broader pH range. The inventors havedetermined that the disulfide linkage contained within2,2'-dithio-bis-ethane sulfonate is stable in a variety of aqueousenvironments (pure water or saline solutions) over a broad pH range,from pH 1.5 to 9.0. One preferred embodiment is a pH of2,2'-dithio-bis-ethane sulfonate ranging from 4 to 9. Lyophilizedformulations containing 2,2'-dithio-bis-ethane sulfonate alone or withcisplatin are stable for longer periods of time (months to years) thantheir aqueous counterparts. When cisplatin is formulated with2,2'-dithio-bis-ethane sulfonate, this invention teaches that theaddition of sufficiently high concentrations of NaCl or HCl arenecessary because the stability of cisplatin is proportionally relatedto the chloride ion concentration of the solution.

One object of this invention is novel compositions which contain bothcis-diammine dichloro platinum and 2,2'-dithio-bis-ethane sulfonate inthe same composition, wherein said composition is provided as an aqueouscomposition or a lyophilized composition which are intended for use inhuman subjects with cancer. The lyophilized compositions containing2,2'-dithio-bis-ethane sulfonate with or without cisplatin and chloridesalts, acids or buffers are reconstituted with a pharmaceuticallyacceptable diluent prior to administration to human subjects withcancer. Examples of pharmaceutically acceptable aqueous diluents includeSterile Water for Injection USP, Dextrose 5% and Water for InjectionUSP, 0.9% NaCl for Injection USP, Dextrose 5% 0.9% NaCl for InjectionUSP, Lactated Ringer's Solution, and the like. Suitable acids for theinstant invention can be selected from the group consisting ofhydrochloric acid and phosphoric acid. Suitable buffers for the instantinvention include acetate and phosphate buffers, which are used whendesired to maintain the solution at a desired pH range.

DEFINITIONS

For the purpose of this invention, certain words and phrases are definedas follows:

"Stable" means that the solution will not undergo major (>1%) chemicalconversion within a reasonable period of time (dependent upon the finalpH of the formulation mixture).

The word "about" when used for pH, cisplatin concentration,2,2'-dithio-bis-ethane sulfonate concentration, NaCl concentration, ormannitol concentration is defined as +/-1%.

"Approximately" is defined to include a range of plus or minus 1%

There are numerous "anticancer agents" but the inventors prefer thefollowing to be used with the claimed invention: 5-FU, bleomycin, VP-16(etoposide), doxorubicin, cyclophosphamide, ifosfamide, trophosphamide,leucovorin, taxol, methotrexate, vincristine and vinblastine.

"Amber vial" is any vial which protects the contents from exposure tofluorescent light.

"Aqueous formulations" is defined as medicaments and pharmaceuticalpreparations which are dissolved or suspended in water and are suitablefor human use.

"Routes of administration" is defined to include parenteral drugadministration (which includes intravenous, intraarterial,intraperitoneal, subcutaneous, intracavitary, intrapleural) and oraldrug administration.

"Injectable" is defined to mean administered parenterally by hypodermicneedle, cannula or catheter (which includes intravenous, intraarterial,intraperitoneal, subcutaneous, intracavitary,and intrapleuraladministration).

"Lyophilized", "lyophilize", "cryodessicated", "reconstituted","reconstitute" or "reconstitutable" are used to describe certaincompositions or formulations and are well known terms of art. Alyophilized composition or formulation in this invention must bereconstituted with aqueous diluents prior to administration.

"pharmaceutically acceptable salts" of 2,2'-dithio-bis-ethane sulfonatefor purposes of this invention include: (a) disodium2,2'-dithio-bis-ethane sulfonate, (b) monosodium 2,2'-dithio-bis-ethanesulfonate, (c) sodium potassium 2,2'-dithio-bis-ethane sulfonate, (d)dipotassium 2,2'-dithio-bis-ethane sulfonate, (e) calcium2,2'-dithio-bis-ethane sulfonate, (f) magnesium 2,2'-dithio-bis-ethanesulfonate and (g) monopotassium 2,2'-dithio-bis-ethane sulfonate. Any ofthese salts may be used in the present invention, but the disodium,monosodium, calcium and magnesium salts are more preferred. When othersalts of 2,2'-dithio-bis-ethane sulfonate are used in place of thedisodium salt, the concentration or proportion of the sulfonate will beadjusted according to molarity. 1 g of the disodium salt,2,2'-dithio-bis-ethane sulfonate is equivalent to 4.63 millimoles of2,2'-dithio-bis-ethane sulfonate anion.

"Simultaneous", "simultaneously", "concurrent", and "concurrently" aredefined as the administration of 2,2'-dithio-bis-ethane sulfonate andcisplatin from either: (1) a composition containing both drugs or (2)separate compositions containing either cisplatin or2,2'-dithio-bis-ethane sulfonate commencing and concluding at the sametime to a patient.

"Preceding" is defined as the administration of 2,2'-dithio-bis-ethanesulfonate within 24 hours before the commencement of administration of adose of cisplatin.

"Following" is defined as the administration of 2,2'-dithio-bis-ethanesulfonate within 24 hours after the conclusion of the administration ofa dose of cisplatin.

"Composition" is defined as a composition of matter.

"Formulation" is defined as a pharmaceutical preparation which containsa mixture of various excipients and key ingredients which provide arelatively stable, desirable and useful form of a drug. For thisinvention, "formulation" is included within the meaning of the term"composition".

"Substantially contemporaneous" is defined as the administration ofcompositions containing 2,2'-dithio-bis-ethane sulfonate as a keyingredient which are administered to the patient who will be or has beentreated with cisplatin within 24 hours; the composition containing2,2'-dithio-bis-ethane sulfonate is administered within 24 hours priorto, simultaneously and/or 24 hours following the administration of thesubsequent component parts. For this invention, "shortly" means within24 hours of the administration of cisplatin. Thus, as an example, acomposition containing 2,2'-dithio-bis-ethane sulfonate formulation isadministered at time X and cisplatin is administered to the patientwithin time X+24 hours. Another example would be the administration of2,2'-dithio-bis-ethane sulfonate one hour before the administration ofcisplatin, followed by the simultaneous administration of2,2'-dithio-bis-ethane sulfonate and cisplatin, which is followed by theadministration of 2,2'-dithio-bis-ethane sulfonate one hour after thecompletion of cisplatin administration in the same patient.

"Cisplatin", "cis-diammine dichloro platinum", and "CDDP" are usedinterchangeably.

"Injectable formulations" and injectable solutions" are usedinterchangeably.

"2,2'-dithio-bis-ethane sulfonate" is defined to include2,2'-dithio-bis-ethane sulfonate and pharmaceutically acceptable saltsof 2,2'-dithio-bis-ethane sulfonate (including, without limitation,dimesna).

"Dimesna" and "disodium 2,2'-dithio-bis-ethane suffonate" refer to thedisodium salt of and "2,2'-dithio-bis-ethane sulfonate".

"Solution" a liquid which may or may not additionally contain dissolvedchemical species. Examples of solutions include water, 0.9% NaCl inwater for injection, dextrose 5%. and water for injection, and the like.

"Untreated or previously treated with an anticancer agent" is defined asa patient diagnosed with cancer who has not received treatment(untreated) or who has received treatment (previously treated) withcytotoxic or hormonal drug therapy directed at curing or palliatingtheir malignancy or their malignant disease.

Aqueous Compositions Containing 2.2'-Dithio-Bis-Ethane Sulfonate

This invention teaches novel compositions containing a water solubledisulfide, 2,2'-dithio-bis-ethane sulfonate as a key ingredient, whereinthe presence of the disulfide and the parenteral administration of thecomposition reduces the risk of cisplatin induced nephrotoxicity,myelosuppression, neurotoxicity and potentiates the antitumor activityof cisplatin when treating human patients with cancer. This inventionalso teaches novel, aqueous parenteral compositions containingcis-diammine dichloro platinum and the water soluble disulfide2,2'-dithio-bis-ethane sulfonate for the purpose of treating patientswith cancer and preventing, retarding the development of, and reducingthe risk of developing cisplatin induced toxicities includingnephrotoxicity, myelosuppression and myelosuppression in human patientswith cancer. Another purpose of this invention is the use of2,2'-dithio-bis-ethane sulfonate to potentiate the antitumor activity ofcisplatin in human patients with cancer. This invention also teachesmethods of preparation and use of aqueous compositions containing2,2'-dithio-bis-ethane sulfonate alone. Additionally, this inventionteaches the addition of certain ingredients to improve the utility ofsaid aqueous compositions as desired.

One object of this invention is a stable aqueous solution whichcomprises 2,2'-dithio-bis-ethane sulfonate with or without any of thefollowing ingredients, in any combination, and without restriction:cisplatin; chloride salts wherein the salt is sodium chloride; andpharmaceutical buffers selected from a group consisting of sodiumacetate and/or phosphate; and hydrochloric acid. All of theseingredients are packaged in a unit dosage form in a sealed container.This composition is suitable for intravenous or intraarterialadministration to human patients with cancer by the injection thereoffrom the container. The following desirable concentration ranges areincluded in the claimed injectable solution: the concentration ofcis-diammine dichloro platinum is between about 0.1 mg/ml and about 1.0mg/ml, the concentration of 2,2'-dithio-bis-ethane sulfonate is between1 mg per ml and about 320 mg/ml, the concentration of sodium chloride isbetween 0.1% and 2.5% by weight of water, and the amounts of buffer(sodium acetate or phosphate) and hydrochloric acid are in an amountsufficient to maintain the pH in the range of 2.0 to 6.0. Anotherembodiment of this invention is the addition of mannitol in aconcentration between about 1.0% to about 2.5% by weight of water to theabove disclosed injectable solutions.

Furthermore, these injectable compositions can be given to a humanpatient with cancer who has not been treated with an anticancer agent(untreated) or this injectable solution can be given to a patient whohas previously been treated or exposed to an anticancer agent(s). Also,this injectable solution can be administered to human patients withcancer in combination with another anticancer agent or agents. There arenumerous "anticancer agents" but the inventors prefer the following tobe used with the claimed invention: 5-FU, bleomycin, VP-16 (etoposide),doxorubicin, cyclophosphamide, ifosfamide, leucovorin, taxol,methotrexate, vincristine and vinblastine.

Aqueous Compositions Containing 2,2'-Dithio-Bis-Ethane Sulfonate Can BeComprised Of:

(i) 2,2'-dithio-bis-ethane sulfonate and water.

(ii) 2,2'-dithio-bis-ethane sulfonate, NaCl, and water.

(iii) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, and water.

(iv) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, phosphoric acid,and water.

(v) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, hydrochloricacid, and water.

(vi) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, phosphoric acid,a buffer, and water.

(vii) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, hydrochloricacid, a buffer, and water.

(viii) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, phosphoricacid, mannitol and water.

(ix) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, hydrochloricacid, mannitol and water.

(x) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, phosphoric acid,mannitol, a buffer and water.

(xi) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, hydrochloricacid, mannitol, a buffer and water.

(xii) 2,2'-dithio-bis-ethane sulfonate, NaCl, hydrochloric acid, andwater.

(xiii) 2,2'-dithio-bis-ethane sulfonate, NaCl, phosphoric acid, andwater.

(xiv) 2,2'-dithio-bis-ethane sulfonate, NaCl, a buffer, phosphoric acidand water.

(xv) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl, a buffer,hydrochloric acid, and water.

Compositions of Lyophilized Compositions Containing2,2'-Dithio-Bis-Ethane Sulfonate as the Key Ingredient of the InstantInvention:

This invention is also useful because lyophilized 2,2'-dithio-bis-ethanesulfonate with or without cisplatin would be a method of choice becauseof the extended shelf life of the lyophilized composition. Additionally,a lyophilized composition is less expensive to manufacture than itsaqueous counterpart. When needed for patient administration, thelyophilized drug composition is reconstituted with an aqueous diluent toyield specific concentrations of 2,2'-dithio-bis-ethane sulfonate aloneor 2,2'-dithio-bis-ethane sulfonate with cisplatin and chloride salts inthe final solution. Reconstitution of lyophilized compositions of2,2'-dithio-bis-ethane sulfonate with or without cisplatin can beachieved by many types of aqueous solutions including, withoutrestriction, 0.9% NaCl USP, Sterile Water for Injection USP, Dextrose 5%and Water for Injection USP, and the like. The inventors have determinedthat the disulfide linkage contained within 2,2'-dithio-bis-ethanesulfonate is stable in a variety of aqueous environments (pure water orsaline solutions) over a broad pH range, from pH 1.5 to 9.0. Onepreferred embodiment is a pH of 2,2'-dithio-bis-ethane sulfonate rangingfrom 4 to 9. The reconstituted solution can be passed through a 0.2micron filter to avoid micro aggregates of the drug product.

This invention teaches novel lyophilized compositions containing a watersoluble disulfide, 2,2'-dithio-bis-ethane sulfonate as a key ingredient,wherein the presence of the disulfide and the parenteral administrationof the formulation reduces the risk of cisplatin induced nephrotoxicity,myelosuppression, and neurotoxicity and also potentiates the antitumoractivity of cisplatin when treating human patients with cancer.

Lyophilized compositions containing 2,2'-dithio-bis-ethane sulfonate asthe key ingredient of the instant invention can be comprised of:

(i) 2,2'-dithio-bis-ethane sulfonate.

(ii) 2,2'-dithio-bis-ethane sulfonate sodium salt.

(iii) 2,2'-dithio-bis-ethane sulfonate, cisplatin and NaCl.

(iv) 2,2'-dithio-bis-ethane sulfonate, cisplatin, NaCl and phosphoricacid.

(v) 2,2'-dithio-bis-ethane sulfonate, cisplatin, mannitol and NaCl.

(vi) 2,2'-dithio-bis-ethane sulfonate, cisplatin, mannitol, NaCl andphosphoric acid.

(vii) 2,2'-dithio-bis-ethane sulfonate and mannitol.

(viii) 2,2'-dithio-bis-ethane sulfonate, mannitol and phosphoric acid.

(ix) 2,2'-dithio-bis-ethane sulfonate, NaCl and phosphoric acid.

(x) 2,2'-dithio-bis-ethane sulfonate, mannitol and hydrochloric acid.

(xi) 2,2'-dithio-bis-ethane sulfonate, NaCl, mannitol and hydrochloricacid.

For this invention the 2,2'-dithio-bis-ethane sulfonate alone or incombination with cisplatin and the other ingredients may be lyophilizedand reconstituted before administration. For the purposes of thisinvention "lyophilized", lyophilize", "freeze-dried", "cryodessicated"all are terms of art which involve the extraction of water from the drugpreparation by evaporation of water from the product. For the purposesof this invention the terms "reconstitute", "reconstituted", or"reconstitutable" are used interchangeably and mean to add apharmaceutically acceptable aqueous diluent to the lyophilizedcomposition to result in an aqueous composition that can be administeredto a human patient with cancer.

Another object of this invention is the administration of saidlyophilized compositions containing 2,2'-dithio-bis-ethane sulfonatewith or without cisplatin and chloride salts, acid or buffer, whereinsaid compositions are used to reduce the risk of cisplatin inducednephrotoxicity when treating human patients with cancer.

Oral Compositions Containing 2,2'-Dithio-Bis-Ethane-Sulfonate

Oral compositions of 2,2'-dithio-bis-ethane sulfonate orpharmaceutically acceptable salts of 2,2'-dithio-bis-ethane sulfonatecan be administered orally to human subjects with cancer who arereceiving cisplatin therapy. Solid dosage forms for oral administrationinclude capsules, tablets, pills, powders and granules. In such soliddosage forms, 2,2'-dithio-bis-ethane sulfonate or a pharmaceuticallyacceptable salt of 2,2'-dithio-bis-ethane sulfonate is admixed with atleast one inert customary excipient (or carrier) such as sodium citrateor dicalcium phosphate or (i) fillers or extenders, as for example,starches, lactose, sucrose, glucose, mannitol and silicic acid, (ii)binders, as for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose and acacia, (iii) humectants, as forexample, glycerol, (iv) disintegrating agents, as for example,agar-agar, calcium carbonate, potato or tapioca starch, align acid,certain complex silicates and sodium carbonate, (v) solution retarders,as for example, paraffin, (vi) absorption accelerators, as for example,quaternary ammonium compounds, (vii) wetting agents, as for example,cetyl alcohol and glycerol monostearate, (viii) adsorbents, as forexample, kaolin and bentonite, and (ix) lubricants, as for example,talc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate or mixtures thereof. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using excipients such as lactoseor milk sugar as well as high molecular weight polyethylene glycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in this art. They may contain opacifyingagents, and can also be of such composition that they release2,2'-dithio-bis-ethane sulfonate or pharmaceutically acceptable salts of2,2'-dithio-bis-ethane sulfonate in a certain part of the intestinaltract in a delayed manner. Examples of embedding compositions which canbe used are polymeric substances and waxes.

The active compounds can also be in microencapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include physiologicallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to 2,2'-dithio-bis-ethane sulfonate or pharmaceuticallyacceptable salts of 2,2'-dithio-bis-ethane sulfonate, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, dimethylacetamide, N-methyl pyrrolidinone, oils, inparticular, cottonseed oil, groundnut oil, corn germ oil, olive oil,polyoxyethylated castor oil, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan or mixtures of theses substances, and the like.

An aspect of the claimed invention is a composition including2,2'-dithio-bis-ethane sulfonate, or a pharmaceutically acceptable saltthereof, cis-diammine dichloro platinum, a chloride salt, wherein thesalt is sodium chloride, and an acid selected from the group consistingof hydrochloric acid and phosphoric acid, in a unit dosage form in asealed container, wherein the composition is suitable for administrationto a human patient with cancer, wherein the concentration ofcis-diammine dichloro platinum is between about 0.1 mg/ml and about 1.0mg/ml, wherein the concentration of 2,2'-dithio-bis-ethane sulfonate isbetween 1.0 mg per ml to about 320 mg per ml, wherein the concentrationof the chloride salt is between 0.1% and 2.5% by weight of water, andwherein the acid is in an amount sufficient to maintain the pH in therange of 2.0 to 6.0. An embodiment of this aspect is where thiscomposition further contains mannitol in a concentration between about1.0% to about 2.5% by weight of water. Yet another embodiment is thiscomposition further contains a sufficient amount of buffer selected fromthe group consisting of sodium acetate and phosphate, alone or incombination, to maintain the pH of the composition in the range of 2.0to 6.0.

Another aspect of this invention is where the human patient with canceris untreated or previously treated with an anticancer agent.

Yet another aspect of this invention is where the composition isadministered in combination with another anticancer agent to the humanpatient with cancer.

A further aspect of this invention is a composition including: a)cis-diammine dichloro platinum in a concentration between about 0.1 mgper ml and about 1.0 mg per ml; b) 2,2'-dithio-bis-ethane sulfonate, ora pharmaceutically acceptable salt thereof, in a concentration betweenabout 1.0 mg per ml and about 320 mg per ml; c) a chloride salt, whereinthe salt is sodium chloride, at a concentration of 0.1% to 2.5% byweight of water; d) mannitol in a concentration between about 1.0% toabout 2.5% by weight of water: and e) acid selected from a groupconsisting of hydrochloric acid and phosphoric acid, alone or incombination, in a sufficient concentration to maintain the pH in therange of 2.0 to 6.0; all of which are in a unit dosage form in a sealedcontainer, wherein the composition is suitable for administration to thehuman patient with cancer. An embodiment of this aspect is where thecomposition further contains a buffer selected from the group consistingof sodium acetate or phosphate, alone or in combination, in a sufficientamount to maintain the pH in the range of 2.0 to 6.0.

Another aspect of this invention is a composition including2,2'-dithio-bis-ethane sulfonate, or a pharmaceutically acceptable saltthereof, and cis-diammine dichloro platinum, in a unit dosage form in asealed container, wherein the composition is suitable for administrationto a human patient with cancer, wherein the concentration of2,2'-dithio-bis-ethane sulfonate is between 1.0 mg per ml to about 320mg per ml and the concentration of cis-diammine dichloro platinum isbetween about 0.1 mg per ml to about 1.0 mg per ml.

Another aspect of the instant invention is a composition including2,2'-dithio-bis-ethane sulfonate, or a pharmaceutically acceptable saltthereof, in a concentration between about 1.0 mg per ml and about 320 mgper ml and this composition may further contain a chloride salt, whereinthe salt is sodium chloride at a concentration of 0.1% to 2.5% by weightof water.

A further aspect of this invention is the above 2,2'-dithio-bis-ethanesulfonate compositions having a pH in the range of 4.0 to 9.0, whereinthe pH range is achieved by adding hydrochloric acid or phosphoric acid.

A further aspect of any of the above compositions wherein thecomposition is in a unit dosage form in a sealed container, wherein thecomposition is suitable for parenteral administration to an untreated orpreviously treated human patient with cancer by the injection orinfusion thereof from the container.

Yet another aspect of any of the above compositions wherein thecomposition is administered in combination with another anticancer agentor agents to a human patient with cancer.

Another aspect of this invention is a lyophilized composition including2,2'-dithio-bis-ethane sulfonate, or a pharmaceutically acceptable saltthereof, wherein the lyophilized composition is reconstituted foradministration preceding, simultaneously with or following theadministration of an anticancer agent or agents to a human patient withcancer.

Yet a further aspect of this invention is a lyophilized compositionincluding 2,2'-dithio-bis-ethane sulfonate, or a pharmaceuticallyacceptable salt thereof, wherein the lyophilized composition isreconstituted to an aqueous parenteral composition prior toadministration, with a pharmaceutically acceptable aqueous diluent toyield an aqueous composition, wherein the concentration of compositioncontains between about 1.0 mg/ml to about 320 mg/ml of2,2'-dithio-bis-ethane sulfonate.

A further aspect of this invention is where the above compositions arereconstituted with a pharmaceutically acceptable aqueous diluentcontaining 0.1% NaCl to about 2.5% NaCl by weight of water to yield aconcentration of 2,2'-dithio-bis-ethane sulfonate between about 1.0mg/ml and about 320 mg/ml.

Another aspect of this invention is a one of the above compositionsfurther contains a sufficient amount of buffer selected from the groupconsisting of sodium acetate and phosphate, or a combination thereof, tomaintain the pH between about 4.0 and 9.0.

An additional aspect of this invention is a lyophilized compositionincluding 2,2'-dithio-bis-ethane sulfonate, or a pharmaceuticallyacceptable salt thereof, and cis-diammine dichloro platinum, wherein thecomposition is reconstituted for administration to a human patient withcancer.

Yet a further aspect of this invention is a lyophilized compositionincluding cis-diammine dichloro platinum, 2,2'-dithio-bis-ethanesulfonate, or a pharmaceutically acceptable salt thereof, and a chloridesalt, wherein the salt is sodium chloride, wherein the composition isreconstituted to an aqueous parenteral composition prior to intravenousadministration with a pharmaceutically acceptable aqueous diluentpreviously acidified to a pH in the range of 2.0 to 6.0 by the additionof sufficient amounts of acid selected from the group consisting ofhydrochloric acid and phosphoric acid, to yield a composition containingcis-diammine dichloro platinum in a concentration between about 0.1mg/ml and about 1.0 mg/ml, wherein the concentration of2,2'-dithio-bis-ethane sulfonate is between about 1.0 mg/ml and about320 mg/ml, wherein the concentration of chloride salt is between 0.1%and 2.5% by weight of water, and wherein the acid is in an amountsufficient to maintain the pH in the range of about 2.0 to about 6.0,and wherein the pharmaceutically acceptable diluent is selected from thegroup consisting of Sterile Water for Injection USP, Dextrose 5% andWater for Injection USP, 0.9% NaCl for Injection USP, Dextrose 5% 0.9%NaCl for Injection USP, and Lactated Ringer's Solution.

An embodiment of this aspect is where the above lyophilized compositionfurther contains a buffer selected from the group consisting of sodiumacetate and phosphate.

Another aspect of this invention is a lyophilized composition including2,2'-dithio-bis-ethane sulfonate, or a pharmaceutically acceptable saltthereof, and a chloride salt, wherein the salt is sodium chloride,wherein the composition is reconstituted to yield a composition foradministration to human subjects with cancer, wherein the concentrationof 2,2'-dithio-bis-ethane sulfonate is between about 1.0 mg per ml andabout 320 mg per ml, wherein the concentration of the chloride salt isbetween 0.1% and 2.5% by weight of water, and wherein the acid is in anamount sufficient to maintain the pH in the range of about 2.0 to about6.0.

Further embodiments of this composition contains a buffer selected fromthe group consisting of sodium acetate and phosphate or mannitol orphosphoric acid.

Another aspect of this invention is a method of administration of2,2'-dithio-bis-ethane sulfonate or pharmaceutically acceptable saltsthereof, wherein aqueous 2,2'-dithio-bis-ethane sulfonate or the saltsare parenterally administered preceding, simultaneously with orfollowing the administration of cis-diammine dichloro platinum to ahuman patient with cancer.

Yet another aspect of this invention is a method of administration of2,2'-dithio-bis-ethane sulfonate, or pharmaceutically acceptable saltsthereof, wherein aqueous 2,2'-dithio-bis-ethane sulfonate or the saltsare orally administered preceding, simultaneously with or following theadministration of cis-diammine dichloro platinum to a human patient withcancer.

Another aspect of this invention is where pharmaceutically acceptablesalt of 2,2'-dithio-bis-ethane sulfonate is selected from the groupconsisting of (a) disodium 2,2'-dithio-bis-ethane sulfonate, (b)monosodium 2,2'-dithio-bis-ethane sulfonate, (c) sodium potassium2,2'-dithio-bis-ethane sulfonate, (d) dipotassium 2,2'-dithio-bis-ethanesulfonate, (e) calcium 2,2'-dithio-bis-ethane sulfonate, (f) magnesium2,2'-dithio-bis-ethane sulfonate and (g) monopotassium2,2'-dithio-bis-ethane sulfonate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Serum Creatinine Values on Day 5 Post Treatment with i.v.Cisplatin (6 mg/kg) With or Without Increasing Doses of i.v.2,2'-Dithio-Bis-Ethane Sulfonate (BNP7787).

FIG. 2. Serum BUN (Blood Urea Nitrogen) Values on Day 5 Post Treatmentwith i.v. Cisplatin (6 mg/kg) With or Without Increasing Doses of i.v.2,2'-Dithio-Bis-Ethane Sulfonate (BNP7787).

FIG. 3. Total WBC Values on Day 5 Post Treatment with i.v. Cisplatin (6mg/kg) With or Without Increasing Doses of i.v. 2,2'-Dithio-Bis-EthaneSulfonate (BNP7787).

FIG. 4. Weight Change in Tumor Bearing Fischer Rats Treated withParenteral Cisplatin (6 mg/kg or 9 mg/kg) and 2,2'-Dithio-Bis-EthaneSulfonate (BNP7787 @ 1,000 mg/kg).

FIG. 5. Antitumor Response of Tumor Bearing Fischer Rats Treated withParenteral Cisplatin (6 mg/kg or 9 mg/kg) and 2,2'-Dithio-Bis-EthaneSulfonate (BNP7787 @ 1000 mg/kg).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In its preferred embodiments, this invention involves the preparationand administration of 2,2'-dithio-bis-ethane sulfonate compositions orformulations preceding, simultaneously with, or following theadministration of cis-diammine dichloro platinum.

EXAMPLES

The following examples illustrate selected modes for carrying out theclaimed invention and are not meant to be construed as limiting thespecification and claims in any way.

Example 1

Preparation of 2.2'-Dithio-Bis-Ethane Sulfonate

2,2'-Dithio-bis-ethane sulfonate was prepared by oxidizing 2-mercaptoethane sulfonate in water with equimolar amount of iodine as previouslyreported by Lamaire and Reiger (Lemaire and Reiger, J. Org. Chem., 26,1330-1, 1961).

Example 2

Stability of 2,2'-Dithio-Bis-Ethane Sulfonate

The stability of 2,2'-dithio-bis-ethane sulfonate at room temperaturewas determined at pH ranges of 1.5 to 9.0. 2,2'-dithio-bis-ethanesulfonate, as synthesized by the method described above, was found to bestable in the pH range of 1.0-11.0.

The stability of 2,2'-dithio-bis-ethane sulfonate in acidic and basicaqueous media can be studied as described in this example. In a typicalexperiment, 50 mg of 2,2'-dithio-bis-ethane sulfonate (as produced byusing the above described method) was dissolved in one ml of water andthe pH of the solution adjusted to 1.5, 2.0, 3.0, 4.0, 5.0 and 6.0 byadding 1N hydrochloric acid in water or the pH adjusted to 8.0, 9.0,10.0 or 11.0 by adding 1N sodium hydroxide in water. The reactionmixture was then stirred for 24 hours at room temperature, the water wasremoved at reduced pressure, dissolved in spectral grade D₂ O, and theproton NMR spectrum was recorded. The results demonstrated that onlypeaks corresponding to the starting material were observed by NMRspectra, and that no additional peaks were observed. These data indicatethat 2,2'-dithio-bis-ethane sulfonate was stable in alkaline or acidicaqueous solutions at pH values from 1.5 to 9.0.

The stability of 2,2'-dithio-bis-ethane sulfonate at pH 1.5 is furtherstudied by heating the reaction mixture to 100 degrees Celsius for 10minutes. No change in the proton spectrum was observed by heating the2,2'-dithio-bis-ethane sulfonate (pH 1.5). These data indicate that2,2'-dithio-bis-ethane sulfonate was stable in alkaline or acidicaqueous solutions at pH values from 1.5 to 9.0.

Example 3

Method #1 to Produce a Formulation Containing Cisplatin and2.2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail one method to produce a formulationcontaining cisplatin and disodium 2,2'-dithio-bis-ethane sulfonate. Forthe purpose of this example, cisplatin and cis-diammine dichloroplatinum are used interchangeably. For the purpose of this example,"approximately" is defined to include a range of plus or minus 1%.

Step 1. USP grade of sodium chloride (NaCl; purchased from VWRScientific) is dissolved in sterile, injectable water to a finalconcentration of 0.9% NaCl by weight of water. A suitable amount of purehydrochloric acid (HCl, 99.999%; purchased from Aldrich ChemicalCompany) is added to the sterile, injectable 0.9% sodium chloridesolution in order to obtain a final pH in the range of approximately 2.0to 6.0.

Step 2. One part by weight of pure, cisplatin (99.999%, purchased fromAldrich Chemical Company) is added to the admixture of Step 1. Thecisplatin is allowed to completely dissolve by agitation (1500-2500 rpm)at room temperature, for approximately 60 to 90 minutes in the dark.

Step 3. Then, 15 parts by weight of disodium 2,2'-dithio-bis-ethanesulfonate (as produced above in Example 1) is added the mixture of Step2. This mixture is agitated until complete dissolution and the final pHis adjusted to a pH ranging between approximately pH 2.0 and pH 6.0 byadding pure hydrochloric acid (99.999%, purchased from Aldrich ChemicalCompany).

Step 4. The solution of Step 3 is sterilized via filtration through asterile 0.2 micron filter (obtained from VWR Scientific).

Step 5. The formulation of Step 4 is stored in sterile injection vialswherein each vial contains approximately 0.9 mg of cisplatin and 14.3 mgof 2,2'-dithio-bis-ethane sulfonate per ml of solution.

Example 4

Method #2 to Produce a Formulation Containing Cisplatin and2.2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail another method to produce aformulation containing cisplatin and disodium 2,2'-dithio-bis-ethanesulfonate. For the purpose of this example, cisplatin and cis-diamminedichloro platinum are used interchangeably. For the purpose of thisexample, "approximately" is defined to include a range of plus or minus1%.

Step 1. USP grade of sodium chloride (NaCl; purchased from VWRScientific) is dissolved in sterile, injectable water to a finalconcentration of 0.9% NaCl by weight of water.

Step 2. Disodium 2,2'-dithio-bis-ethane sulfonate (as produced above inExample 1; fifteen mg per milliliter of solution is added to thesterile, injectable 0.9% NaCl solution from Step 1. The2,2'-dithio-bis-ethane sulfonate is allowed to completely dissolve byagitation (1500-2500 rpm) at room temperature. Complete dissolutionrequires approximately 5-10 minutes at room temperature. The pH of the2,2'-dithio-bis-ethane sulfonate solution is adjusted to a pH rangingbetween approximately pH 2.0 and pH 6.0 by adding pure (99.999% purity)hydrochloric acid.

Step 3. Pure (99.999% purity) cisplatin is added (1 part by weight) tothe solution of Step 2. This mixture is agitated until completedissolution and the final pH is adjusted to a pH ranging betweenapproximately pH 2.0 and pH 6.0 by adding pure (99.999% purity)hydrochloric acid.

Step 4. The solution of Step 3 is sterilized via filtration through asterile 0.2 micron filter (obtained from VWR Scientific).

Step 5. The formulation of Step 4 is stored in sterile injection vialswherein each vial contains approximately 0.9 mg of cisplatin and 14.3 mgof 2,2'-dithio-bis-ethane sulfonate per ml of formulation.

Example 5

Method #3 to Produce a Formulation Containing Cisplatin and2.2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail another method to produce aformulation containing cisplatin and disodium 2,2'-dithio-bis-ethanesulfonate. For the purpose of this example, cisplatin and cis-diamminedichloro platinum are used interchangeably. For the purpose of thisexample, "approximately" is defined to include a range of plus or minus1%.

Step 1. A suitable amount of pure, disodium 2,2'-dithio-bis-ethanesulfonate (as produced in Example 1) is dissolved in sterile, injectablewater to a concentration of 15.0 mg/ml.

Step 2. USP grade sodium chloride crystals (NaCl; purchased from VWRScientific) is added to the solution of Step 1 such that the finalconcentration of NaCl is 0.9% by weight of water.

Step 3. The pH of the 2,2'-dithio-bis-ethane sulfonate--NaCl solution ofStep 2 is adjusted to range between approximately pH 2.0 and pH 6.0 bythe addition of pure (99.999% purity), hydrochloric acid (purchased fromAldrich Chemical Company).

Step 4. An amount of pure (99.999% purity) cisplatin is added to thesolution of Step 3 such that the final concentration is approximately1.0 mg/ml cisplatin and the solution is agitated until completedissolution of the cisplatin is achieved. The dissolution of cisplatinin the solution is carried out in the dark.

Step 5. The solution of Step 4 is sterilized via filtration through asterile 0.2 micron filter.

Step 6. The formulation of Step 5 is store in sterile injection vialswherein each vial contains approximately 1.0 mg of cisplatin and 14.3 mgof 2,2'-dithio-bis-ethane sulfonate per ml of injection solution.

Example 6

Method #4 to Produce a Formulation Containing Cisplatin and2.2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail another method to produce aformulation containing cisplatin and disodium 2,2'-dithio-bis-ethanesulfonate. For the purpose of this example, cisplatin and cis-diamminedichloro platinum are used interchangeably. For the purpose of thisexample, "approximately" is defined to include a range of plus or minus1%.

Step 1. USP grade sodium chloride (NaCl; purchased from VWR Scientific)dissolved in sterile, injectable water to a final concentration of 0.9%NaCl by weight of water.

Step 2. The pH of this NaCl solution is brought to approximately 2.0 to6.0 by the addition of 99.999% pure hydrochloric acid (purchased fromAldrich Chemical Company).

Step 3. Pure (99.999% purity) cisplatin (obtained from Aldrich ChemicalCompany) is added in 0.5 mg to each milliliter of the solution obtainedin Step 2 and allowed to dissolve completely by agitation (1500-2500rpm) for approximately 60 to 90 minutes at room temperature in the dark.

Step 4. Then, 30 milligrams of disodium 2,2'-dithio-bis-ethane sulfonate(as produced in Example 1) is added to each milliliter of the solutionStep 3. The 2,2'-dithio-bis-ethane sulfonate--cisplatin mixture isallowed to completely dissolve with agitation at room temperature.

Step 5. The pH of the disodium 2,2'-dithio-bis-ethanesulfonate--cisplatin solution is adjusted to a final pH ranging betweenapproximately 2.0 and 6.0 by the addition of pure (99.999% purity)hydrochloric acid (obtained from Aldrich Chemical Company).

Step 6. The solution of Step 5 is sterilized via filtration through asterile 0.2 micron filter (obtained from VWR Scientific).

Step 7. The formulation of Step 6 is stored in sterile injection vialswherein each vial contains 0.5 mg of cisplatin and 30.0 mg of disodium2,2'-dithio-bis-ethane sulfonate per ml of injection solution.

Example 7

Method #5 to Produce a Formulation Containing Cisplatin and2.2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail another method to produce aformulation containing cisplatin and disodium 2,2'-dithio-bis-ethanesulfonate. For the purpose of this example, cisplatin and cis-diamminedichloro platinum are used interchangeably. For the purpose of thisexample, "approximately" is defined to include a range of plus or minus1%.

Step 1. USP grade sodium chloride (NaCl; purchased from VWR Scientific)dissolved in sterile, injectable water to a final concentration of 0.9%NaCl by weight of water.

Step 2. An amount of pure (99.999% purity) hydrochloric acid (obtainedfrom Aldrich Chemical Company) is added to the NaCl solution of Step 1in order to obtain a final pH in the range of approximately 2.0 to 6.0.

Step 3. An amount of USP grade potassium chloride crystals (KCl;purchased from VWR Scientific) is dissolved in the solution of Step 2(0.9% NaCl) such that the final concentration of potassium chloride is0.1% by weight.

Step 4. One milligram of pure (99.999% purity) cisplatin is added toeach milliliter of the solution of Step 3 and is completely dissolved byagitation (1500 to 2500 rpm) for approximately 60 to 90 minutes at roomtemperature in the dark.

Step 30 milligrams of disodium 2,2'-dithio-bis-ethane sulfonate (asproduced by Example 1) is added to each milliliter the solution of Step4. This mixture is agitated until complete dissolution and the final pHranging between approximately pH 2.0 and pH 6.0 by adding pure (99.999%purity) hydrochloric acid (purchased from Aldrich Chemical Company).

Step 6. The solution of Step 5 is sterilized via filtration through asterile 0.2 micron filter (obtained from VWR Scientific).

Step 7. The formulation of Step 6 is stored in sterile injection vialswherein each vial contains approximately 1.0 mg of cisplatin and 30.0 mgof disodium 2,2'-dithio-bis-ethane sulfonate per ml of solution.

Example 8

Method #6 to Produce a Formulation Containing Cisplatin and2.2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail another method to produce aformulation containing cisplatin and disodium 2,2'-dithio-bis-ethanesulfonate. For the purpose of this example, cisplatin and cis-diamminedichloro platinum are used interchangeably. For the purpose of thisexample, "approximately" is defined to include a range of plus or minus1%.

Step 1. USP grade sodium chloride (NaCl; purchased from VWR Scientific)dissolved in sterile, injectable water to a final concentration of 0.9%NaCl by weight of water. A suitable amount of pure (99.999% purity)hydrochloric acid is added to the sterile, injectable 0.9% sodiumchloride solution in order to obtain a final pH in the range ofapproximately 2.0 to 6.0.

Step 2. Pure mannitol (99+% purity, purchased from Aldrich ChemicalCompany) is dissolved in the solution of Step 1 so that theconcentration of mannitol in said solution is 1.0% by weight of thesolution.

Step 2. One milligram of pure, cisplatin (purchased from AldrichChemical Company, grade 99.999% purity) is added to each milliliter ofthe solution of Step 2. The cisplatin is allowed to completely dissolveby agitation (1500-2500 rpm) at room temperature. This should takeapproximately 60 to 90 minutes at room temperature in the dark.

Step 4. Then, 30 mg of disodium 2,2'-dithio-bis-ethane sulfonate (asproduced in Example 1) is added to each milliliter of the solution ofStep 3. This solution is agitated until complete dissolution of2,2'-dithio-bis-ethane sulfonate is achieved and the final pH isadjusted to a pH ranging between approximately pH 2.0 and pH 6.0 byadding pure (99.999% purity) hydrochloric acid (purchased from AldrichChemical Company).

Step 5. The solution of Step 4 is sterilized via filtration through asterile 0.2 micron filter.

Step 6. The formulation of Step 5 is stored in sterile injection vialswherein each vial contains approximately 1.0 mg of cisplatin and 30.0 mgof disodium 2,2'-dithio-bis-ethane sulfonate per ml of injectionsolution.

Example 9

Stability of 2,2'-Dithio-Bis-Ethane Sulfonate and Cisplatin Formulations

This example was designed to study the stability of2,2'-dithio-bis-ethane sulfonate and cisplatin formulations.

1. First, 2,2'-dithio-bis-ethane sulfonate--cisplatin formulations wereprepared according to Examples 3 through 8.

2. The final pH of each formulation was adjusted to a range of 2.0 to6.0.

3. Each pH adjusted 2,2'-dithio-bis-ethane sulfonate--cisplatinformulation was stored in a sealed glass vial protected from fluorescentlight at room temperature (approximately 27 plus or minus 2 degreesCelsius).

4. The stability of each pH adjusted 2,2'-dithio-bis-ethanesulfonate--cisplatin formulation was analyzed on a weekly basis for atleast 6 (six) months by nuclear magnetic resonance (NMR) or HPLCanalysis. The NMR spectra or HPLC analysis was compared to a freshlyprepared and pH adjusted 2,2'-dithio-bis-ethane sulfonate--cisplatinformulation. The similarity of NMR spectra or HPLC retention times andpeaks corresponding to the freshly prepared formulation denotesstability of the pH adjusted formulation over time.

Example 10

Method to Produce a Parenteral Solution Containing Cisplatin and2.2'-Dithio-Bis-Ethane Sulfonate from a Lyophilized Formulation ofCisplatin and 2.2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail one method to produce a formulationcontaining cisplatin and disodium 2,2'-dithio-bis-ethane sulfonate byreconstitution of a lyophilized formulation of cisplatin and disodium2,2'-dithio-bis-ethane sulfonate. For the purpose of this example,cisplatin and cis-diammine dichloro platinum are used interchangeably.For the purpose of this example, "approximately" is defined to include arange of plus or minus 1%.

Step 1. USP grade of sodium chloride (NaCl; purchased from VWRScientific) is dissolved in sterile, injectable water to a finalconcentration of 0.9% NaCl by weight of water. A suitable amount of purehydrochloric acid (HCl, 99.999%; purchased from Aldrich ChemicalCompany) is added to the sterile, injectable 0.9% sodium chloridesolution in order to obtain a final pH in the range of approximately 2.0to 6.0.

Step 2. One part by weight of pure, cisplatin (99.999%, purchased fromAldrich Chemical Company) is added to the admixture of Step 1. Thecisplatin is allowed to completely dissolve by agitation (1500-2500 rpm)at room temperature, for approximately 60 to 90 minutes in the dark.

Step 3. Then, 15 parts by weight of disodium 2,2'-dithio-bis-ethanesulfonate (as produced above in Example 1) is added the mixture of Step2. This mixture is agitated until complete dissolution and the final pHis adjusted to a pH ranging between approximately pH 2.0 and pH 6.0 byadding pure hydrochloric acid (99.999%, purchased from Aldrich ChemicalCompany).

Step 4. The acidified NaCl solution containing cisplatin and2,2'-dithio-bis-ethane sulfonate from step 3 is then lyophilized using acommercially available equipment. The lyophilized drug formulation canbe stored at room temperature in amber vials which are shielded fromlight for 6 months to one year until needed for patient administration.

Step 5. When needed for patient administration, the lyophilized drugformulation Step 4 is reconstituted to yield a cisplatin concentrationof 0.9 mg per milliliter and a concentration of 2,2'-dithio-bis-ethanesulfonate of 14.3 mg per milliliter by using Sterile Water USP forInjection which has been acidified to a pH of 2.0 to 6.0 by using asufficient quantity of pure hydrochloric acid or phosphoric acid. Thisreconstituted aqueous formulation is passed through a sterile 0.2 micronfilter and can be administered to the patient.

Example 11

Method to Produce a Parenteral Solution Containing Cisplatin and2,2'-Dithio-Bis-Ethane Sulfonate from a Lyophilized Formulation ofCisplatin and 2,2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail one method to produce a formulationcontaining cisplatin and disodium 2,2'-dithio-bis-ethane sulfonate byreconstitution of a lyophilized formulation of cisplatin and disodium2,2'-dithio-bis-ethane sulfonate. For the purpose of this example,cisplatin and cis-diammine dichloro platinum are used interchangeably.For the purpose of this example, "approximately" is defined to include arange of plus minus 1%.

Step 1. USP grade of sodium chloride (NaCl; purchased from VWRScientific) is dissolved in sterile, injectable water to a finalconcentration of 0.9% NaCl by weight of water. A suitable amount of purephosphoric acid (H3PO4, 99.999%; purchased from Aldrich ChemicalCompany) is added to the sterile, injectable 0.9% sodium chloridesolution in order to obtain a final pH in the range of approximately 2.0to 6.0.

Step 2. One milligram of pure, cisplatin (99.999%, purchased fromAldrich Chemical Company) is added to each milliliter of the solution ofStep 1. The cisplatin is allowed to completely dissolve by agitation(1500-2500 rpm) at room temperature, for approximately 60 to 90 minutesin the dark.

Step 3. Then, 15 milligrams of disodium 2,2'-dithio-bis-ethane sulfonate(as produced above in Example 1) is added to each milliliter of thesolution of Step 2. This solution is agitated until complete dissolutionand the final pH is adjusted to a pH ranging between approximately pH2.0 and pH 6.0 by adding pure hydrochloric acid (99.999%, purchased fromAldrich Chemical Company).

Step 4. The acidified NaCl solution containing cisplatin and2,2'-dithino-bis-ethane sulfonate from step 3 is then lyophilized usinga commercially available equipment. The lyophilized drug formulation canbe stored at room temperature in amber vials which are shielded fromlight for 6 months to one year until needed for patient administration.

Step 5. When needed for patient administration, the lyophilzed drugformulation Step 4 is reconstituted to yield a cisplatin concentrationof approximately 1.0 mg per milliliter and a concentration of2,2'-dithio-bis-ethane sulfonate of 15.0 mg per milliliter by usingSterile Water USP for Injection. This reconstituted aqueous formulationis passed through a sterile 0.2 micron filter (obtained from VWRScientific) and can be administered to the patient.

Example 12

Method to Produce a Parenteral Solution Containing2,2'-Dithio-Bis-Ethane Sulfonate from a Lyophilized Formulation of2,2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail one method to produce a formulationcontaining disodium 2,2-dithio-bis-ethane sulfonate for the purpose ofadministering said solution during human cancer therapy withcis-diammine dichloro platinum by reconstitution of a lyophilizedformulation of disodium 2,2'-dithio-bis-ethane sulfonate. For thepurpose of this example, "approximately" is defined to include a rangeof plus or minus 1%.

Step 1. USP grade of sodium chloride (NaCl; purchased from VWRScientific) is dissolved in Sterile Water for Injection USP to a finalconcentration of 0.9% NaCl by weight of water. Alternatively Sterile0.9% NaCl Solution for Injection may be used for Step 1.

Step 2. Then, 15 milligrams of disodium 2,2'-dithio-bis-ethane sulfonate(as produced above in Step 1) is added to each milliliter of thesolution in Step 1. This mixture is agitated until complete dissolutionof the 2,2'-dithio-bis-ethane sulfonate is observed (30 to 60 minutes).

Step 3. The 0.9% NaCl solution containing dissolved2,2'-dithio-bis-ethane sulfonate from Step 2 is then lyophilized using acommercially available equipment. The lyophilized drug formulationcontaining 2,2'-dithio-bis-ethane sulfonate can be stored at roomtemperature in amber vials which are shielded from light for 6 months toone year until needed for patient administration.

Step 4. When needed for patient administration, the lyophilized2,2'-dithio-bis-ethane sulfonate drug formulation from Step 3 isreconstituted to yield a concentration of 2,2'-dithio-bis-ethanesulfonate of 15.0 mg per milliliter by using Sterile Water USP forInjection. This reconstituted aqueous 2,2'-dithio-bis-ethane sulfonateformulation is passed through a sterile 0.2 micron filter (obtained fromVWR Scientific) and can be administered to the patient.

Example 13

Method to Produce An Aqueous Parenteral Solution Containing2,2'-Dithio-Bis-Ethane Sulfonate

This example was designed to detail one method to produce a formulationcontaining disodium 2,2'-dithio-bis-ethane sulfonate for the purpose ofadministering said solution during human cancer therapy withcis-diammine dichloro platinum. For the purpose of this example,"approximately" is defined to include a range of plus or minus 1%.

Step 1. USP grade of sodium chloride (NaCl; purchased from VWRScientific) is dissolved in Sterile Water for Injection USP to a finalconcentration of 0.9% NaCl by weight of water. Alternatively Sterile0.9% NaCl Solution for Injection may be used for Step 1.

Step 2. Then, 15 mg of disodium 2,2'-dithio-bis-ethane sulfonate (asproduced above in Step 1) is added to each milliliter of the solution inStep 1. This mixture is agitated until complete dissolution of the2,2'-dithio-bis-ethane sulfonate is observed (30 to 60 minutes). Thefinal concentration of disodium 2,2'-dithio-bis-ethane sulfonate is 15mg/ml.

Step 3. When needed for patient administration, the solution containing2,2'-dithio-bis-ethane sulfonate drug formulation from Step 2 is can beadministered to the patient.

Example 14

In Vivo Demonstration of Protection by 2,2'-Dithio-Bis-Ethane Sulfonate(BNP7787) Against Cisplatin Induced Nephrotoxicity and Myelosuppressionin Fischer Rats: Use of Concurrently Administered 2,2'-Dithio-Bis-EthaneSulfonate and Cisplatin by Parenteral Injection

This Example was designed to demonstrate the in vivo protective effectsof 2,2'-dithio-bis-ethane sulfonate (also referred to as BNP7787)administered at 1,000 mg/kg by a single i.v. injection in Fischer rats(150-200 g) receiving a nephrotoxic dose of cisplatin (6 mg/kg i.v.single injection) when the two drugs are administered substantiallycontemporaneously (cisplatin and 2,2'-dithio-bis-ethane sulfonate givenconcurrently). The data from these studies are shown below in FIGS. 1and 2. Under Good Laboratory Practice (GLP) conditions, Fischer rats (10rats per treatment group) were treated with or without escalating dosesof BNP7787 (up to 1000 mg/kg) administered by i.v. injection with anephrotoxic dose of cisplatin (6 mg/kg). Serum BUN (FIG. 2) andCreatinine (FIG. 1) values were measured on day 5 and the animals wereweighed daily. As shown in FIGS. 1 and 2, BNP7787 demonstrates asignificant (100% renal protection at 333 and 1000 mg/kg of BNP7787)renal protective effect as assessed by day 5 Creatinine (FIG. 1) and BUN(FIG. 2) values compared to cisplatin treated controls. At the 37 mg/kgdose of BNP7787, a slight increase in nephrotoxicity (as measured byplasma BUN and creatinine levels and by histopathology) relative tocisplatin only treated groups was observed (FIG. 2). FIG. 3 demonstratesa dose-dependent protective effect of 2,2'-dithio-bis-ethane sulfonateagainst cisplatin induced myelosuppression as measured by mean plasmaWhite Blood Counts (WBC). The mean day 5 WBC levels of rats treated withhigher doses of 2,2'-dithio-bis-ethane sulfonate, namely the 333 mg/kgand 1,000 mg/kg treatment groups, are approximately 6,800 and 6,200,respectively. These mean WBC values are within 15% of the WBC values foruntreated controls. The day 5 mean WBC counts for the cisplatin only andlow dose (37 mg/kg) 2,2'-dithio-bis-ethane sulfonate treatment groupsare approximately 4,800 and 4,500, respectively and represent a 32% to36% reduction in the day 5 mean WBC from the untreated control group.The Fischer rat model is highly correlated with cisplatin inducednephrotoxicity in humans and is thus an excellent animal model tosupport the utility of 2,2'-dithio-bis-ethane sulfonate to protectagainst cisplatin induced nephrotoxicity in humans. This study documentsseveral aspects of the invention: (1) evidences a clear dose-responseeffect of 2,2'-dithio-bis-ethane sulfonate in providing renal protectionfrom cisplatin induced nephrotoxicity, (2) evidences the ability of2,2'-dithio-bis-ethane sulfonate to provide 100% renal protection from ahighly nephrotoxic dose of cisplatin, and (3) evidences a dose dependentprotective effect of 2,2'-dithio-bis-ethane sulfonate against cisplatininduced myelosuppression.

Example 15

Potentiation of Cisplatin Antitumor Activity by ParenteralAdministration of BNP7787 (2,2'-dithio-bis-ethane sulfonate)

The antitumor activity and toxicity, as measured by weight changesestimated by changes in tumor volumes and animal weights, respectively,of escalating doses of parenterally (intravenously) administeredcisplatin (6 mg/kg and 9 mg/kg) with or without substantiallycontemporaneously intravenously administered BNP7787 (1000 mg/kg) wasinvestigated in Fischer rats bearing subcutaneous established (˜3.0 g)WARD colon cancer. In the untreated control rats, it should be notedthat the subcutaneous 3.0 gram WARD colon carcinoma tumors will growfrom 3.0 grams to about 10 grams in size in about 7 days (shown in FIG.5). In FIG. 4, the data show that untreated tumor bearing rats and ratstreated with BNP7787 only lose about 2 to 4% of their body weight inabout 6 days (open and closed circles, respectively). It is notable thatrats treated with cisplatin alone (6 mg/kg and 9 mg/kg) lose up to 8% oftheir body weight at 6 days. Treatment with i.v. BNP7787 (1000 mg/kg) atboth dose levels of cisplatin (6 mg/kg and 9 mg/kg) were clearlyprotective against renal damage as measured by weight loss, and the ratsin these groups receiving cisplatin and BNP7787 demonstrated greatermean weights than all other treatment groups (compare open and closedsquares to open and closed triangles). This observation suggests thattreatment with BNP7787 may prevent or reduce other cisplatin toxicities,including neurotoxicity and emesis, leading to weight loss in rats.Another important observation is that the cisplatin/BNP7787 treatedgroups had potentiation of cisplatin antitumor activity for both the 6mg/kg and 9 mg/kg dose groups (FIG. 5-open and closed squares,respectively). Rats treated with cisplatin only at doses of 6 mg/kg and9 mg/kg had a maximum reduction in median tumor weight from 3,000 mg to700 and 500 mg, respectively. Rats treated with cisplatin at doses of 6mg/kg and 9 mg/kg immediately followed by ad seriatim administration ofBNP7787 using a single i.v. dose of 1,000 mg/kg had a maximum reductionin median tumor weight from 3,000 mg to less than 300 and 100 mg,respectively. This data in FIG. 5 evidences several important featuresof 2,2'-dithio-bis-ethane sulfonate when it is used substantiallycontemporaneously with cisplatin: (1) 2,2'-dithio-bis-ethane sulfonatelacks intrinsic antitumor activity, (2) substantially contemporaneouslyadministered 2,2'-dithio-bis-ethane sulfonate does not abrogate theantitumor activity of cisplatin, (3) substantially contemporaneousadministration of 2,2'-dithio-bis-ethane sulfonate appears to potentiatethe antitumor activity of cisplatin and (4) these data demonstrate thesignificant antitumor potentiation of cisplatin antitumor activity ismediated and enhanced by 2,2'-dithio-bis-ethane sulfonate.

Example 16

Stability of 2,2'-dithio-bis-ethane sulfonate At pH Ranging From 1.5 to9

A control solution of the disodium salt of 2,2'-dithio-bis-ethanesulfonate was prepared by dissolving 9 mg of sodium chloride (USP grade)and 30 mg of the disodium salt of 2,2'-dithio-bis-ethane sulfonate in 1ml of deuterated water (99.999%). The pH of the control was adjusted to7.0 using 1N DC1. This deuterated solution was directly used as thecontrol for the NMR sample.

Preparation of the assay solutions containing 2,2'-dithio-bis-ethanesulfonate were also made in a similar manner, 30 mg of the disodium saltof 2,2'-dithio-bis-ethane sulfonate was added to each vial containing 1ml of 0.9% sodium chloride in deuterated water and agitated to dissolvecompletely. The homogenous solution obtained was then adjusted to thedesired pH using either 1N DC1 or 1N deuterated sodium hydroxidesolution. The pH adjusted aqueous solutions were then analyzed by highfield NMR (300 MHz, Bruker) for a period of 48 hours at three hourintervals. The stability of the disodium salt of 2,2'-dithio-bis-ethanesulfonate in aqueous solution at pH 1.5 was also determined at elevatedtemperature by heating the aqueous solution at 100° C. for 15 minutes.

¹ H-NMR spectra of the monosodium salt of 2-mercapto ethane sulfonatesodium (mesna), and the disodium salt of 2,2'-dithio-bis-ethanesulfonate provided two sets of distinctive peaks whereby molecularinterconversions or degradation could be monitored. The proton NMRspectrum of mesna provided multiplets at 2.88δ and 3.19δ whereas thedisodium salt of 2,2'-dithio-bis-ethane sulfonate compound hadcharacteristic multiplet signals at 3.09δ.

Example 17

Oral Formulations Containing 2,2'-Dithio-Bis-Ethane-Sulfonate

Oral formulations of 2,2'-dithio-bis-ethane sulfonate orpharmaceutically acceptable salts of 2,2'-dithio-bis-ethane sulfonatecan be administered orally to human subjects with cancer who arereceiving cisplatin therapy. Solid dosage forms for oral administrationinclude capsules, tablets, pills, powders and granules. In such soliddosage forms, 2,2'-dithio-bis-ethane sulfonate or a pharmaceuticallyacceptable salt of 2,2'-dithio-bis-ethane sulfonate is admixed with atleast one inert customary excipient (or carrier) such as sodium citrateor dicalcium phosphate or (i) fillers or extenders, as for example,starches, lactose, sucrose, glucose, mannitol and silicic acid, (ii)binders, as for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose and acacia, (iii) humectants, as forexample, glycerol, (iv) disintegrating agents, as for example,agar-agar, calcium carbonate, potato or tapioca starch, align acid,certain complex silicates and sodium carbonate, (v) solution retarders,as for example, paraffin, (vi) absorption accelerators, as for example,quaternary ammonium compounds, (vii) wetting agents, as for example,cetyl alcohol and glycerol monostearate, (viii) adsorbents, as forexample, kaolin and bentonite, and (ix) lubricants, as for example,talc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate or mixtures thereof. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.

Example 18

Solubility of 2,2'-Dithio-Bis-Ethane Sulfonate In Sterile Water

In this example, 10 grams of the disodium salt of 2,2'-dithio-bis-ethanesulfonate was dissolved in 50 ml of sterile HPLC grade water and the pHof the solution was readjusted to a pH of 7.0 using 1N hydrochloricacid. The resulting solution was then passed through a 0.2 micronsyringe filter into an autoclaved round bottom flask (250 ml). Thissterile solution was then freeze dried (lyophilized) to a whitecrystalline powder. The lyophilized powder was then used for thefollowing solubility studies.

1 ml of sterile HPLC grade water was distributed in each of tensterilized 10 ml vials. As described in FIG. 6 below, varying amounts ofthe lyophilized disodium salt of 2,2'-dithio-bis-ethane sulfonate werethen added to each vial. Each vial was first observed at roomtemperature for a period of approximately 15 minutes, and then heated toa temperature of 37° C. for a period of at least 15 minutes. The Tablebelow demonstrates that, based on the tyndall effect observed in eachvial, 2,2'-dithio-bis-ethane sulfonate is soluble in concentrationsranging from about 10.5 mg per ml to about 102 mg per ml.

    ______________________________________    Solubility of 2,2'-dithio-bis-ethane sulfonate    (BNPI 7787) in sterile water at 37° C.                   Vol. of          Qty. of  Sterile    Entry BNPI     water   Incubation                                   Observation                                           Observation    No.   7787 (mg)                   (ml)    time (min.)                                   at 25° C.                                           at 37° C.    ______________________________________    1      10.50   1       15      Clear   Clear    2      20.50   1       15      Clear   Clear    3      45.50   1       15      Clear   Clear    4      51.65   1       15      Clear   Clear    5      69.13   1       15      Slight  Clear                                   yndall    6      94.33   1       15      Tyndall Clear    7      98.63   1       15      Tyndall Clear after                                           30 min.    8     102.83   1       15      Tyndall Clear after                                           360 min.    ______________________________________

Example 19

Solubility of 2,2'-Dithio-Bis-Ethane Sulfonate in Sterile Water and 0.9%Sodium Chloride Solution

For this example, 10 grams of the disodium salt of2,2'-dithio-bis-ethaiie sultonate BNPI7787) was dissolved in 50 ml ofsterile HPLC grade water and the pH of the solution was readjusted to apH of 7.15 using 1N hydrochloric acid. The resulting solution was thenpassed through a 0.2 micron syringe filter into an autoclaved roundbottom flask (250 ml). This sterile solution was then freeze dried(lyophilized) to a white crystalline powder. The lyophilized powder wasthen used for the following solubility studies.

1 ml of sterile HPLC grade water was distributed in each of one set ofsix sterilized 10 ml vials. 1 ml of 0.9% sodium chloride solution wasthen distributed in each of a second set of six sterilized 10 ml vials.The 0.9% sodium chloride solution was obtained by dissolving 9 mg of USPgrade sodium chloride in sterile HPLC grade water using an agitator. Asdescribed in the table below, varying amounts of the lyophilizeddisodium salt of 2,2'-dithio-bis-ethane sulfonate were then added toeach of the 12 vials. Each vial was first observed at room temperaturefor a period of approximately 15 minutes and then heated to atemperature of 37° C. for a period of at least 15 minutes. The tablebelow demonstrates that, based on the tyndall effect observed in eachvial, 2,2'-dithio-bis-ethane sulfonate is soluble in sterile water andin sodium chloride solution in concentrations of 2,2'-dithio-bis-ethanesulfonate ranging from about 100 mg per ml to about 320 mg per ml.

    ______________________________________    Solubility of 2,2'-dithio-bis-ethane sulfonate    in sterile water and NaCl solution at 37° C. & pH 7.15         Qty. of  Sterile    Entry         BNPI     water   0.9% NaCl                                  Observation                                           Observation    No.  7787 (mg)                  (ml)    soln. (ml)                                  at 25° C.                                           at 37° C.    ______________________________________    1    100      1       0       Clear    Clear    2    100      0       1       Clear    Clear    3    200      1       0       Clear    Clear    4    200      0       1       Clear    Clear    5    300      1       0       Clear    Clear    6    300      0       1       Clear    Clear    7    320      1       0       Clear    Clear    8    320      0       1       Slight tydall                                           Clear    9    350      1       0       Not clear                                           Clear    10   350      0       1       Not Clear                                           Not clear    11   400      1       0       Not Clear                                           Not clear    12   400      0       1       Not Clear                                           Not clear    ______________________________________

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U.S. Pat. No. 4,310,515, entitled "Pharmaceutical Compositions ofCisplatin," Issued Jan. 12, 1982.

The foregoing description has been directed to particular embodiments ofthe invention in accordance with the requirements of the Patent Statuesfor the purposes of illustration and explanation. It will be apparent,however, to those skilled in this art, that many modifications, changes,and variations in the claimed solutions and methods set forth will bepossible without departing from the scope and spirit of the claimedinvention. It is intended that the following claims be interpreted toembrace all such modifications, changes, and variations.

What is claimed is:
 1. A pharmaceutical formulation comprising2,2'-dithio-bis-ethane sulfonate, or a pharmaceutically acceptable saltthereof, and cis-diammine dichloro platinum, wherein said pharmaceuticalformulation is in lyophilized form and is dissolved prior toadministration to a human patient with cancer.
 2. A pharmaceuticalformulation comprising cis-diammine dichloro platinum, lyophilized2,2'-dithio-bis-ethane sulfonate, or a pharmaceutically acceptable saltthereof, and sodium chloride, wherein said cis-diammine dichloroplatinum and lyophilized 2,2'-dithio-bis-ethane sulfonate or apharmaceutically acceptable salt thereof are dissolved prior tointravenous administration with a pharmaceutically acceptable aqueousdiluent at a pH range of 2.0 to 6.0.
 3. The lyophilized composition ofclaim 2 further containing a buffer selected from the group consistingof sodium acetate and phosphate.